U.S. patent application number 16/347091 was filed with the patent office on 2019-09-05 for method for preparing fermentable sugars from lignocellulosic biomass.
This patent application is currently assigned to Inbicon A/S. The applicant listed for this patent is Inbicon A/S. Invention is credited to Anna Frederike Gossmann, Anna Granly Hansen, Martin Dan Jeppesen, Jan Larsen, Kit Haubjerg Mogensen, Hanne Risbjerg Sorensen, Laila Thirup, Lars Villadsgaard Toft.
Application Number | 20190271017 16/347091 |
Document ID | / |
Family ID | 60812010 |
Filed Date | 2019-09-05 |
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United States Patent
Application |
20190271017 |
Kind Code |
A1 |
Jeppesen; Martin Dan ; et
al. |
September 5, 2019 |
METHOD FOR PREPARING FERMENTABLE SUGARS FROM LIGNOCELLULOSIC
BIOMASS
Abstract
A method for providing a C5/C6 product from a lignocellulosic
material is disclosed, said method comprising the steps: (i)
pretreatment of the lignocellulosic material; (ii) solid/liquid
separation of the pretreated lignocellulosic material from step (a)
into a first solid fraction and a first liquid fraction; (iii)
enzymatic fiber hydrolysis of said first solid fraction from step
(b) by use of an enzyme composition capable of degrading
lignocellulosic material, thereby providing a C5/C6 fiber slurry
comprising C5 and/or C6 sugars; (iv) solid/liquid separation of the
C5/C6 fiber slurry from step (c) into a second solid fraction and a
second liquid fraction; and optionally (v) combining said first
liquid fraction and said second liquid fraction for enzymatic mixed
sugar hydrolysis (MSH), whereby a MSH C5/C6 product is
provided.
Inventors: |
Jeppesen; Martin Dan;
(Odense V, DK) ; Larsen; Jan; (Tommerup, DK)
; Mogensen; Kit Haubjerg; (Fredericia, DK) ;
Gossmann; Anna Frederike; (Flensburg, DK) ; Hansen;
Anna Granly; (Fredericia, DK) ; Thirup; Laila;
(Skanderborg, DK) ; Toft; Lars Villadsgaard;
(Silkeborg, DK) ; Sorensen; Hanne Risbjerg;
(Holte, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Inbicon A/S |
Fredericia |
|
DK |
|
|
Assignee: |
Inbicon A/S
Fredericia
DK
|
Family ID: |
60812010 |
Appl. No.: |
16/347091 |
Filed: |
November 6, 2017 |
PCT Filed: |
November 6, 2017 |
PCT NO: |
PCT/EP2017/078340 |
371 Date: |
May 2, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62417570 |
Nov 4, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08L 95/00 20130101;
C12P 7/10 20130101; Y02E 50/16 20130101; C07G 1/00 20130101; C13K
1/02 20130101; C12P 2203/00 20130101; Y02E 50/10 20130101; C12P
2201/00 20130101; C13K 13/002 20130101; C12P 19/02 20130101; C12P
19/14 20130101 |
International
Class: |
C12P 19/02 20060101
C12P019/02; C12P 19/14 20060101 C12P019/14; C13K 1/02 20060101
C13K001/02; C12P 7/10 20060101 C12P007/10; C07G 1/00 20060101
C07G001/00; C08L 95/00 20060101 C08L095/00 |
Claims
1. A method for providing a C5/C6 product from a lignocellulosic
material, such as soft lignocellulosic biomass, comprising the
steps: a) Pretreatment of the lignocellulosic material; b)
Solid/liquid separation of the pretreated lignocellulosic material
from step (a) into a first solid fraction and a first liquid
fraction; c) Enzymatic fiber hydrolysis of said first solid
fraction from step (b) by use of an enzyme composition capable of
degrading lignocellulosic material, thereby providing a C5/C6 fiber
slurry comprising C5 and/or C6 sugars; d) Solid/liquid separation
of the C5/C6 fiber slurry from step (c) into a second solid
fraction and a second liquid fraction; and optionally e) Combining
said first liquid fraction and said second liquid fraction for
enzymatic mixed sugar hydrolysis (MSH), whereby a MSH C5/C6 product
is provided; and optionally. f) Enzymatic fiber cake hydrolysis of
said second solid fraction from step (d) to obtain a slurry C5/C6
product.
2. The method according to claim 1, wherein the pretreatment is
conducted: a. at a dry matter (DM) content in the range of 5-80%,
such as 10-70%, such as 20-60%, or such as 30-50%, or at a DM
content around 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, or 80% or at a DM content of more than 80%;
and/or b. at low, medium, or high severity; and/or at conditions
providing a xylan number of >10%, 6-10% or <6%.
3. The method according to claim 1 or 2, wherein the enzymatic
fiber hydrolysis, fiber cake hydrolysis and/or MSH is/are
conducted: a. for a period of at least 6 h, 12 h, 24 h, 48 h, or 72
h, such as 6-120 h, 12-100 h, or 48-96 h, or around 12 h, 24 h, 48
h, 72 h, 96 h, or 120h; and/or b. at a pH in the range of at least
pH 3.0, such as pH 3.0-6.0, such as pH 4.0-5.5, such as pH 4.2-5.4,
and/or around a pH of 4.2, 4.5, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3
or 5.4; and/or c. at a temperature in the range of 30-70.degree.
C., 40-65.degree. C., 50-62.degree. C., or 55-60.degree. C., and/or
around 40.degree. C., 42.degree. C., 44.degree. C., 46.degree. C.,
48.degree. C., 50.degree. C., 52.degree. C., 54.degree. C.,
56.degree. C., 58.degree. C., 60.degree. C., 62.degree. C.,
64.degree. C., 66.degree. C., 68.degree. C., or 70.degree. C.;
and/or a DM content of at least 10%, such as around 15-45%, 20-40%,
25-35%, and/or at a DM content of around 15%, 20%, 25%, 30%, 35%,
or 40 %.
4. The method according to any one of the preceding claims, wherein
the enzyme composition capable of degrading lignocellulosic
material comprises: a. a cellulase and/or a hemicellulose; and/or
b. a mixture of cellulase(s) and/or hemicellulase(s); and/or c. one
or more of xylanase(s), xylosidase(s), arabinoxylanase(s),
xyloglucanase(s), glucoronoxylanase(s), glucomannanase(s),
esterase(s) and any combination thereof; and optionally wherein the
esterases comprise one or more acetylesterases and/or feroyl
esterases; and/or one or more of endocellulase(s),
endoglucanase(s), exocellulase(s), exoglucanase(s),
endoxylanase(s), acetyl xylan esterase(s), xylosidase(s),
.beta.-glucosidase(s) and any combination thereof.
5. The method according to any of the preceding claims, wherein
step (e) is conducted by combining said first liquid fraction and
said second liquid fraction and enzymatically hydrolysing the
mixture.
6. The method according to claim 4 or 5, wherein hemicellulase(s)
is/are also present in step (e); and optionally wherein the
hemicellulase(s) present in step (e) comprise(s) xylanase(s),
xylosidase(s), arabinoxylanase(s), xyloglucanase(s),
glucoronoxylanase(s), glucomannanase(s), esterase(s),
acetylesterases, feroyl esterase(s) and any combination
thereof.
7. The method according to any one of claims 4-6, wherein all or at
least a fraction of the hemicellulase(s) present in step (e) has
been added in step (c); and/or one or more hemicellulase(s) is/are
added in step (e); and/or wherein one or more additional enzyme(s)
are added in step (e), and optionally wherein the additional
enzyme(s) are essentially not present in the enzyme composition
capable of degrading lignocellulosic material added in step (c);
and/or the additional enzyme(s) is one or more of:
hemicellulase(s), xylanase(s), xylosidase(s), arabinoxylanase(s),
xyloglucanase(s), glucoronoxylanase(s), glucomannanase(s),
esterase(s), acetylesterases, feroyl esterase(s), and any
combination thereof.
8. The method according to any of the preceding claims, wherein
step (e) comprises an ultrafiltration step (j) for recycling
enzymes present after MSH, such as an ultrafiltration step adapted
to allow for recycling of at least 30% (w/w), 50% (w/w), 75% (w/w),
80% (w/w), or 90% (w/w) of the enzyme activity.
9. The method according to any one of claims 4-8, wherein
cellulase(s) is/are also present in step (f), and optionally
wherein all or a fraction of the cellulase(s) present in step (f)
has been added in step (c).
10. The method according to any one of claims 4-9, wherein one or
more hemicellulase(s) is/are added in step (f).
11. The method according to any one of the preceding claims,
wherein the MSH and/or fiber cake hydrolysis are performed without
addition of one or more enzyme(s), such as without addition of one
or more cellulase(s) and/or one or more hemicellulase(s).
12. The method according to any one of the preceding claims,
further comprising the step(s) of: (g) Solid/liquid separation of
the slurry C5/C6 product from step (f) into a third solid fraction
and a liquid C5/C6 product; and/or (k) Combining at least a portion
of the MSH C5/C6 product with at least a portion of one or more of:
the slurry C5/C6 product from step (f), the liquid C5/C6 product
from step (g), and/or the second liquid fraction from step (d) to
obtain a combined C5/C6 product.
13. The method according to any one of the preceding claims,
wherein a. the second liquid fraction possesses a lower inhibitor
concentration than the first liquid fraction; b. the second liquid
fraction possesses a lower inhibitor concentration than the MSH
C5/C6 product; and/or c. the slurry C5/C6 product possesses a lower
inhibitor concentration than the MSH C5/C6 product.
14. The method according to any one of the preceding claims,
wherein the combined C5/C6 product consists or consists essentially
of the MSH C5/C6 product and the slurry C5/C6 product from step
(f); the MSH C5/C6 product and the liquid C5/C6 product from step
(g); or the MSH C5/C6 product and the second liquid fraction from
step (d).
15. The method according to any one of the preceding claims,
further comprising a lignin recovery step, such as removal of
water, compacting and/or pelleting, and optionally, wherein said
lignin recovery is conducted on the second or third solid fraction
provided in steps (d) or (g).
16. A method for providing a fermentation product, said method
comprising the steps of: m) Providing at least one C5/C6 product
according to the method of any one of the preceding claims; and n)
Providing the fermentation product by a fermentation of said C5/C6
product with a microorganism; and optionally, wherein the C5/C6
product comprises one or more: MSH C5/C6 product, Slurry C5/C6
product, Liquid C5/C6 product, Combined C5/C6 product, first liquid
fraction, or second liquid fraction, and any combination
thereof.
17. The method according to any one of the preceding claims,
wherein a fermentation product is provided in a fermentation broth,
said method further comprising the step(s) of: (o) Recovering said
fermentation product from a fermentation broth and/or (p)
Recovering lignin from a spent fermentation broth, and/or a
fraction provided in steps (n) or (o).
18. The method according to any one of the preceding claims,
wherein a fermentation is carried out in at least a first and a
second fermentation step, wherein a first and a second fermentation
substrate are fermented.
19. A two-step fermentation method comprising the steps of: aa)
Pretreatment of the lignocellulosic material; bb) Solid/liquid
separation of the pretreated lignocellulosic material from step
(aa) into a first solid fraction and a first liquid fraction; cc)
Enzymatic fiber hydrolysis of said first solid fraction from step
(bb) by use of an enzyme composition capable of degrading
lignocellulosic material, thereby providing a C5/C6 fiber slurry;
dd) Solid/liquid separation of the C5/C6 fiber slurry from step
(cc) into a second solid fraction and a second liquid fraction; ee)
Enzymatic mixed sugar hydrolysis (MSH) of a mixture of the first
liquid fraction from step (bb) and the C5/C6 fiber slurry from step
(cc), or the first liquid fraction from step (bb) and the second
liquid fraction from step (dd), thereby providing a C5/C6 MSH
product; ff) Providing a first fermentation substrate comprising at
least a portion of the C5/C6 fiber slurry and/or the second liquid
fraction; gg) Providing a second fermentation substrate comprising
at least a portion of the C5/C6 MSH product; hh) Fermenting the
first fermentation substrate in a first fermentation with a
microorganism; and ii) Fermenting the second fermentation substrate
in a subsequent second fermentation; wherein step (dd) is optional;
and optionally wherein the first fermentation substrate possesses a
significantly lower inhibitor concentration than the second
fermentation substrate; and/or the first fermentation is a batch or
fed-batch fermentation.
20. The method according to claim 18 or 19, wherein the first
fermentation is carried out by providing a first fermentation
substrate comprising: a. the second liquid fraction provided in
step (d) or (dd); b. the C5/C6 fiber slurry provided in step (c) or
(cc); and/or a. the C5/C6 product obtained in step (f), i.e. the
liquid C5/C6 product or the slurry C5/C6 product; and optionally,
wherein the first fermentation is carried out by providing a first
fermentation substrate consisting essentially of: a. the second
liquid fraction provided in step (d) or (dd); b. the C5/C6 fiber
slurry provided in step (c) or (cc); and/or c. the C5/C6 product
obtained in step (f), i.e. the liquid C5/C6 product or the slurry
C5/C6 product.
21. The method according to any one of claims 18-20, wherein the
first fermentation substrate comprises or consists essentially of a
mixture of the second liquid fraction and the C5/C6 product
obtained in step (f), i.e. the liquid C5/C6 product or the slurry
C5/C6 product, and optionally, wherein the ratio between the second
liquid fraction and the C5/C6 product is in the range of
100:0.1-0.1:100 (w/w), such as 10:0.1-0.1:10 (w/w), or such as
10:1-1:10 (w/w); or around 50:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1,
7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,
1:8, 1:9, 1:10, 1:15, 1:20, 1:25, or 1:50 (w/w).
22. The method according to any one of claims 18-21, wherein a. the
first fermentation substrate is provided essentially without
dilution with process water; and/or b. the second fermentation is a
fed-batch fermentation or a continuous fermentation, optionally
conducted in the same fermenter as the first fermentation; and/or
c. the fed-batch fermentation is with linear or exponential feed;
and/or d. the second fermentation is conducted with the same
microorganisms as in the first fermentation; and/or the second
fermentation is carried out by providing a second fermentation
substrate comprising or consisting essentially of a mixture of the
C5/C6 product obtained in step (f) (i.e. the liquid C5/C6 product
or slurry C5/C6 product) and the C5/C6 product obtained from step
(e) (i.e. MSH C5/C6 product); and optionally, wherein the ratio
between the liquid C5/C6 product or slurry C5/C6 product and the
C5/C6 product obtained from step (e) (i.e. MSH C5/C6 product) is in
the range of 100:0.1-0.1:100 (w/w), such as 10:0.1-0.1:10 (w/w),
such as 10:1-1:10 (w/w), such as 5:1-1:5 (w/w); such as 4:1-1:4
(w/w), such as 3:1-1:3 (w/w), such as 2.5-1:2.5 (w/w), such as
2:1-1:2 (w/w) or such as 1.5-1:1-1.5 (w/w); or around 50:1, 25:1,
20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1,
1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15,
1:20, 1:25, 1:50 (w/w); and/or e. the second fermentation is
carried out by providing a second fermentation substrate comprising
or consisting essentially of the C5/C6 MSH product provided in step
(ee); and/or f. the second fermentation is provided essentially
without dilution with process water; and/or g. the volume of the
first fermentation is significantly smaller than the volume of the
second fermentation, and optionally, wherein the volume of the
first fermentation is 2-40%, 3-30%, 5-20%, 7.5-15%, 8-12%, or
around 10% of the volume of the second fermentation; and/or h.
wherein the fermentation product is recovered, such as by
distillation; and/or i. said method comprises a lignin recovery
step, such as from a distillation remnant.
23. The method according to any one of claims 18-22, wherein the
first and second fermentation are consecutive fermentations,
optionally conducted in the same fermenter; and/or wherein the
second fermentation comprises fermentation of both the first liquid
fraction and the C5/C6 fiber slurry.
24. The method according to any one of claims 16-23, wherein the
fermentation product is an alcohol, organic acid, vitamin, amino
acid, peptide, enzyme, and/or a C1-C4 product, such as one or more
of: methanol, ethanol, butanol, acetone, formic acid, acetic acid,
propionic acid, butyric acid, oxalic acid, lactic acid, malic aid,
and any combination thereof.
25. The method according to any one of claims 16-24, wherein the
microorganism is a eukaryotic or prokaryotic microorganism capable
of fermenting C5 and C6 sugars, such as xylose and glucose, such as
a bacterium or a yeast, such as Saccharomyces cerevisiae, capable
of or adapted to fermenting xylose and glucose to EtOH; and
optionally, wherein the microorganism is a recombinant
microorganism.
26. A method for preparing ethanol and optionally lignin from a
lignocellulosic material comprising the steps of: Providing at
least one C5/C6 product according to a method according to any one
of the preceding claims; Fermentation of said at least one C5/C6
product to convert sugars to ethanol in the fermentation broth with
a yeast; Isolation of an ethanol-rich fraction from the
fermentation broth; and optionally Isolation of lignin; and
optionally wherein the fermentation is conducted according to a
method according to any one of the preceding claims.
27. Lignin provided from lignocellulosic biomass according to any
one of the preceding claims.
28. A C5/C6 product provided according to any one of the preceding
claims.
29. A fermentation substrate comprising a C5/C6 product provided by
a method according to any one of the preceding claims.
30. The first or second fermentation substrate provided by a method
according to any one of the preceding claims.
31. Use of lignin according to claim 27 in a bitumen composition,
such as asphalt.
32. A composition comprising 0.1-99.9% (w/w) lignin according to
claim 27.
33. A bitumen composition comprising: a. 1-99.89% (w/w) bitumen; b.
0.1-50% (w/w) lignin according to claim 27; c. 0.01-20% (w/w)
plasticity modifying agent(s); and d. 0-95% (w/w) further
component(s); and optionally wherein the plasticity modifying agent
is one or more plastomer, one or more thermoplastic elastomer, one
or more rubber, one or more viscosity modifier, and/or one or more
reactive polymer, including any combination thereof; and/or wherein
the further component(s) is one or more dispersing agent(s),
surfactant(s), hydrotropic agent(s), emulsifier(s), preserving
agent(s), anti-foaming agent (s), viscosity modifier(s), reactive
polymer(s) and any combination thereof; and/or one or more
aggregate(s) and/or filler(s), such natural, manufactured, recycled
aggregates, including any combination thereof.
34. Use of a composition according to claim 32 or 33 in sealing
work, road work, paving work, providing a surface layer, providing
a sealing layer, providing a road and providing a pavement,
providing a top layer of a road; and/or in one or more applications
relating to (i) agriculture, (ii) buildings and industrial paving,
(iii) hydraulics and erosion control, (iv) industrial, (v) paving,
(vi) railways, and (vii) recreation, such as ad (i) disinfectants,
fence post coating, mulches, mulching paper, paved barn floors,
barnyards, feed platforms, protecting tanks, vats, protection for
concrete structures, tree paints (protective); ad (ii): water and
moisture barriers (above and below ground), floor compositions,
tiles, coverings, insulating fabrics, papers, step treads, building
papers, caulking compounds, cement waterproofing compounds, glass
wool compositions, insulating fabrics, felts, papers, joint filler
compounds, laminated roofing shingles, liquid roof coatings,
plastic cements, shingles, acoustical blocks, compositions, felts,
bricks, damp-proofing coatings, compositions, insulating board,
fabrics, felts, paper, masonry coatings, plasterboards, putty,
soundproofing, stucco base, wallboard, air-drying paints,
varnishes, artificial timber, ebonised timber, insulating paints,
plumbing, pipes, treated awnings, canal linings, sealants; ad
(iii): catchment areas, basins, dam groutings, dam linings,
protection, dyke protection, ditch linings, drainage gutters,
structures, embankment protection, groynes, jetties, levee
protection, mattresses for levee and bank protection, membrane
linings, waterproofing, reservoir linings, revetments, sand dune
stabilisation, sewage lagoons, oxidation ponds, swimming pools,
waste ponds, water barriers, backed felts, ad (iv): conduit
insulation, lamination, insulating boards, paint compositions,
papers, pipe wrapping, insulating felts, panel boards, underseal,
battery boxes, carbons, electrical insulating compounds, papers,
tapes, wire coatings, junction box compound, moulded conduits,
black grease, buffing compounds, cable splicing compound,
embalming, etching compositions, extenders, explosives, lap cement,
plasticisers, preservatives, printing inks, well drilling fluid,
armoured bituminised fabrics, burlap impregnation, mildew
prevention, sawdust, cork, asphalt composition, acid-proof enamels,
mastics, varnishes, acid-resistant coatings, air-drying paints,
varnishes, anti-corrosive and anti-fouling paints, anti-oxidants
and solvents, base for solvent compositions, baking and
heat-resistant enamels, boat-deck sealing compound, lacquers,
japans, marine enamels, blasting fuses, briquette binders, burial
vaults, casting moulds, clay articles, clay pigeons, expansion
joints, flowerpots, foundry cores, friction tape, gaskets, mirror
backing, rubber, moulded compositions, shoe fillers, soles; ad (v):
airport runways, taxiways, aprons, asphalt blocks, brick fillers,
bridge deck, surfacing, crack fillers, floors for buildings,
warehouses, garages, highways, roads, streets, shoulders, kerbs,
gutters, drainage ditches, parking lots, driveways, Portland cement
concrete underseal, roof-deck parking, pavements, footpaths, soil
stabilisation; ad (vi) ballast treatment, dust laying, paved
ballast, sub-ballast, paved crossings, freight yards, station
platforms; and ad (vii) dance pavilions, drive-in movies,
gymnasiums, sport arenas, playgrounds, school yards, race tracks,
running tracks, skating rinks, swimming and wading pools, tennis
courts, handball courts, synthetic playing fields and running track
surfaces.
Description
FIELD
[0001] The present invention relates to a method for processing
lignocellulosic biomass to fermentable sugars, to a method for
producing a fermentation product, such as by the use of a two-step
fermentation method, and to a method for producing lignin.
BACKGROUND
[0002] Historical reliance on petroleum and other fossil fuels has
been associated with dramatic and alarming increases in atmospheric
levels of greenhouse gases. International efforts are underway to
mitigate greenhouse gas accumulation, supported by formal policy
directives in many countries. One central focus of these mitigation
efforts has been the development of processes and technologies for
utilization of renewable plant biomass to replace petroleum as a
source of precursors for fuels and other chemical products.
[0003] Industrial manufacture of fuel ethanol from sugar and
starch-based plant materials, such as sugarcane, root and grain
crops, is already in wide global use. However, both environmental,
economic and moral objections have been raised to these "first
generation" bioethanol processes, e.g. for placing demand for crops
as human food into direct competition with demand for fuel for
personal automobiles.
[0004] Great interest has therefore arisen in developing biomass
conversion systems that do not consume food crops--so-called
"second generation" biorefining, whereby bioethanol and other
products can be produced from lignocellulosic biomass such as crop
wastes (stalks, cobs, pits, stems, shells, husks, etc.), grasses,
straws, wood chips, waste paper and the like. In "second
generation" technology, fermentable 6-carbon (C6) sugars derived
primarily from cellulose and fermentable 5-carbon (C5) sugars
derived from hemicellulose are liberated from biomass
polysaccharide polymer chains by enzymatic hydrolysis or, in some
cases, by pure chemical hydrolysis. The fermentable sugars obtained
from biomass conversion in a "second generation" biorefinery can be
used to produce e.g. ethanol, acetone, butanol, lactic acid, and/or
other compounds useful as e.g. fuel or precursors for chemical
products, e.g. various polymers etc.
[0005] The total yield of both C5 and C6 sugars is a key factor in
the economic viability of commercialization of lignocellulosic
biomass processing. Because of limitations of its physical
structure, lignocellulosic biomass cannot be effectively converted
to fermentable sugars by enzymatic hydrolysis without some
pretreatment process. A wide variety of different pretreatment
schemes have been reported, each offering different advantages and
disadvantages.
[0006] WO2014/019589, herewith incorporated by reference in its
entirety, discloses a method for processing of lignocellulosic
biomass comprising a pretreatment and enzymatic processing of a
solid fraction to produce a C5/C6 product.
[0007] WO2015/014364, herewith incorporated by reference in its
entirety, discloses a method for processing lignocellulosic biomass
using a single-stage autohydrolysis pretreatment and enzymatic
hydrolysis.
BRIEF DESCRIPTION OF FIGURES
[0008] FIG. 1: Schematic outline of process steps pertaining to
methods according to the present invention.
[0009] FIG. 2: Process scheme (1) depicts a process scheme of a
relatively simple process configuration, such as a "whole slurry"
process as described in WO2015/014364.
[0010] FIG. 3: Process scheme (2) depicts a more complex process
scheme comprising a "C5 bypass", also termed "V2" herein, such as
processes described in WO 2014/019589.
[0011] FIG. 4: Process scheme (3) depicts an embodiment/process
scheme according to the current invention (also termed "V2.X" or
"twostep hydrolysis and mixed sugar hydrolysis" herein).
[0012] FIG. 5: Experimental design for comparison of total
carbohydrate conversion in the V2 and V2.X method.
[0013] FIG. 6: Glucan conversion as a function of enzyme dose for
the V2.X, V2 and C5 bypass method with lines to guide the eye.
[0014] FIG. 7: Xylan conversion as a function of enzyme dose for
the V2.X and the C5 by-pass method with lines to guide the eye.
[0015] FIG. 8: Arabinan conversion as a function of enzyme dose for
the V2.X method and C5 by-pass method with lines to guide the
eye.
[0016] FIG. 9: Glucan conversion of fibers for V2.X and the C5
by-pass method for five different pretreatment dates.
[0017] FIG. 10: Xylan conversion of fibers for Version 2.X and the
C5 by-pass method for five different pretreatment dates.
[0018] FIG. 11: Total Glucan conversion: Grey circle--One stage
hydrolysis with 75 g Cellic.RTM. CTec3/kg glucan added in the fiber
hydrolysis (FH) and 22 wt-% SS, Dark grey triangle--One stage
hydrolysis with 75 g Cellic.RTM. CTec3/kg glucan added in the fiber
hydrolysis (FH) and 18 wt-% SS, Light grey cross [0019] Two stage
hydrolysis with 75 g Cellic.RTM. CTec3/kg glucan added in the fiber
hydrolysis (FH) and 22 wt-% SS in the fiber hydrolysis and 22 wt-%
SS in the fiber cake hydrolysis, and Black un-filled circle [0020]
Two stage hydrolysis with 50 g Cellic.RTM. CTec3/kg glucan added in
the fiber hydrolysis (FH) and 22 wt-% SS in the fiber hydrolysis
and 25 g Cellic.RTM. CTec3/kg FH glucan (giving 75 g Cellic.RTM.
CTec3/kg FH glucan in total) added in the fiber cake hydrolysis
(FCH) and 22 wt-% SS in the fiber cake hydrolysis.
[0021] FIG. 12: Xylan conversion in MSH. Colour code dark grey: MSH
including fibers; light grey: MSH, where the fibers are removed
before addition of C5-bypass.
[0022] FIG. 13: Total xylan conversion in MSH as function of enzyme
dose at 48 hours reaction time (0 hours for heat treated with no
enzymes) at pH 5, 250 rpm and 50.degree. C.
[0023] FIG. 14: Embodiment of a V2 setup with improved
fermentation.
[0024] FIG. 15: Schematic outline of a two-step fermentation.
[0025] FIG. 16: Changes in xylan conversion at increasing
proportions of C5 liquid in the post hydrolysis. "Filtrate" may
refer to the liquid fraction after a hydrolysis step, such as
hydrolysis step (c) in FIG. 14.
SUMMARY OF THE INVENTION
[0026] In a first aspect, the current invention pertains to a
method for providing a C5/C6 product from a lignocellulosic
material comprising the steps: [0027] a) Pretreatment of the
lignocellulosic material; [0028] b) Solid/liquid separation of the
pretreated lignocellulosic material from step (a) into a first
solid fraction and a first liquid fraction; [0029] c) Enzymatic
fiber hydrolysis of said first solid fraction from step (b) by use
of an enzyme composition capable of degrading lignocellulosic
material, thereby providing a C5/C6 fiber slurry comprising C5
and/or C6 sugars; [0030] d) Solid/liquid separation of the C5/C6
fiber slurry from step (c) into a second solid fraction and a
second liquid fraction; and optionally [0031] e) Combining said
first liquid fraction and said second liquid fraction for enzymatic
Mixed sugar hydrolysis (MSH), whereby a MSH C5/C6 product is
provided.
[0032] In a second aspect, the current invention relates to a
method for providing a fermentation product, said method comprising
the steps of: [0033] m) Providing at least one C5/C6 product
according to the method of any one of the preceding embodiments;
and [0034] n) Providing the fermentation product by a fermentation
of said C5/C6 product with a microorganism.
[0035] In a third aspect, the current invention concerns a two-step
fermentation method comprising the steps of: [0036] aa)
Pretreatment of the lignocellulosic material; [0037] bb)
Solid/liquid separation of the pretreated lignocellulosic material
from step (aa) into a first solid fraction and a first liquid
fraction; [0038] cc) Enzymatic fiber hydrolysis of said first solid
fraction from step (bb) by use of an enzyme composition capable of
degrading lignocellulosic material, thereby providing a C5/C6 fiber
slurry; [0039] dd) Solid/liquid separation of the C5/C6 fiber
slurry from step (cc) into a second solid fraction and a second
liquid fraction; [0040] ee) Enzymatic mixed sugar hydrolysis (MSH)
of a mixture of the first liquid fraction from step (bb) and the
C5/C6 fiber slurry from step (cc), or the first liquid fraction
from step (bb) and the second liquid fraction from step (dd),
thereby providing a C5/C6 MSH product; [0041] ff) Providing a first
fermentation substrate comprising at least a portion of the "C5/C6
fiber hydrolysis slurry" and/or the second liquid fraction; [0042]
gg) Providing a second fermentation substrate comprising at least a
portion of the C5/C6 MSH product; [0043] hh) Fermenting the first
fermentation substrate in a first fermentation with a
microorganism; and [0044] ii) Fermenting the second fermentation
substrate in a subsequent second fermentation; wherein step (dd) is
optional.
[0045] In a fourth aspect, the current invention concerns a method
for preparing ethanol and lignin from a lignocellulosic material
comprising the steps of: [0046] Providing at least one C5/C6
product according to a method according to any one of the preceding
aspects; [0047] Fermentation of said at least one C5/C6 product to
convert sugars to ethanol in the fermentation broth with a yeast;
[0048] Isolation of an ethanol rich fraction from the fermentation
broth; and optionally [0049] Isolation of lignin.
[0050] In a fifth aspect, the current invention pertains to lignin
provided from lignocellulosic biomass according to any one of the
preceding aspects.
[0051] In a sixth aspect, the current invention relates to a C5/C6
product provided according to any one of the preceding aspects.
[0052] In a seventh aspect, the current invention concerns a
fermentation substrate comprising a C5/C6 product provided by a
method according to any one of the preceding aspects.
[0053] In an eighth aspect, the current invention pertains to a
first or second fermentation substrate provided by a method
according to any one of the preceding aspects.
[0054] In a ninth aspect, the current invention relates to
compositions comprising lignin obtained or obtainable by a method
according to any of the previous aspects, including different uses
of said lignin-comprising compositions.
DETAILED DESCRIPTION
[0055] Methods for preparing hydrolysed lignocellulosic biomass
which can be fermented by microorganisms to yield small organic
molecules are only suited for large-scale industrial use if such
methods are economically competitive methods. In order to be
economically competitive such methods must exhibit high yield of C6
sugars and/or C5 sugars, as low as possible consumption of energy,
enzymes and other prerequisites on the cost-side as well as
preferably provide by-products having a significant value.
[0056] The present inventors have surprisingly found a method for
preparing a C5/C6 and/or a C6+C5 product from a lignocellulosic
material which exhibit relatively low energy input, fast and
efficient hydrolysis of the lignocellulosic material to C6 and/or
C5 sugars, while at the same time providing a substantial amount of
high-value lignin as a by-product.
[0057] The particular advantage of the present invention is that
separate hydrolysis of liquid and solid fractions of pretreated
lignocellulosic biomass produces at least two C5/C6 and/or C6+C5
product fraction, one e.g. having mainly C6 sugars and low
concentration of fermentation inhibitory substances and one e.g.
having mainly C5 sugars and higher amount of fermentation
inhibitory substances. Hence, fermentation of the C5/C6 and/or
C6+C5 products may advantageously be carried out by first
fermenting the low inhibitor fraction and then subsequently adding
and fermenting the high inhibitor fraction.
[0058] In some embodiments, the present invention relates to a
method for preparing at least one C5/C6 product, such as a C6
and/or C5 sugar, from a lignocellulosic material comprising the
steps: [0059] I. Pretreatment of the lignocellulosic material,
[0060] II. Solid/liquid separation into a first solid fraction and
a first liquid fraction, [0061] III. Enzymatic hydrolysis of said
first solid fraction from step II) by use of an enzyme composition
comprising at least one cellulase and/or hemicellulase (such as a
xylanase), [0062] IV. Solid/liquid separation of the reaction
mixture from step III) into a second solid fraction and a second
liquid fraction, and optionally [0063] V. Mixing of said first
liquid fraction and said second liquid fraction for enzymatic
hydrolysis to obtain a C5/C6 product, and optionally [0064] VI.
Recycling enzymes present after enzymatic hydrolysis in step
III).
[0065] FIG. 1 shows different embodiments of the invention, in
particular a method for providing a C5/C6 product from a
lignocellulosic material comprising the steps: [0066] a)
Pretreatment of the lignocellulosic material; [0067] b)
Solid/liquid separation of the pretreated lignocellulosic material
from step (a) into a first solid fraction and a first liquid
fraction; [0068] c) Enzymatic fiber hydrolysis of said first solid
fraction from step (b) by use of an enzyme composition capable of
degrading lignocellulosic material, thereby providing a C5/C6 fiber
hydrolysis slurry comprising C5 and/or C6 sugars; [0069] d)
Solid/liquid separation of the C5/C6 fiber slurry from step (c)
into a second solid fraction and a second liquid fraction; and
optionally [0070] e) Combining said first liquid fraction and said
second liquid fraction for enzymatic Mixed sugar hydrolysis (MSH),
whereby a MSH C5/C6 product is provided; and optionally [0071] f)
Enzymatic fiber cake hydrolysis of said second solid fraction from
step (d) to obtain a slurry C5/C6 product; and optionally [0072] g)
Solid/liquid separation of the slurry C5/C6 product from step (f)
into a third solid fraction and a liquid C5/C6 product; and
optionally [0073] h) Combining at least a portion of the MSH C5/C6
product with at least a portion of one or more of: the slurry C5/C6
product from step (f), the liquid C5/C6 product from step (g),
and/or the second liquid fraction from step (d) to obtain a
combined C5/C6 product; and optionally [0074] i) Ultrafiltration
step for recycling enzymes present after the MSH in step (e).
[0075] The liquid fraction provided by the solid/liquid separations
of steps b) and d) can be maintained separately from the solid
fractions during enzymatic hydrolysis, c.f. FIG. 1. Separate
enzymatic hydrolysis of the solid fractions may take place in the
fiber hydrolysis in step c) and the fiber cake hydrolysis in step
f), thus providing advantages of the current invention in
comparison to the prior art, such as a higher yield of C6 and C5
sugars from the solid fraction and leaving the slurry C6+C5 product
rich in high-value lignin in the solid part. Usually, the liquid
fractions obtained in solid/liquid separation steps b) and d) can
be combined and subjected to a mixed sugar hydrolysis (MSH), such
as disclosed in step e), c.f. FIG. 1.
[0076] As used herein, the following terms have the following
meaning:
[0077] The terms "C5/C6 product" and/or "C6/C5 product" can be used
interchangeably, and is/are meant to comprise a composition
comprising at least one C6 sugar and/or at least one C5 sugar,
where C6 sugar and C5 sugar may be any carbohydrate having six or
five carbon atoms, respectively.
[0078] The terms "C6+C5 product" and/or "C6/C5 product" can be used
interchangeably, and is/are meant to comprise a composition
comprising at least one C6 sugar and at least one C5 sugar, where
C6 sugar and C5 sugar may be any carbohydrate having six or five
carbon atoms, respectively.
[0079] The C5/C6 and/or C6+CS product may be a liquid, a suspension
or slurry, or a solid composition and it may contain additional
compounds in addition to the C6 sugar and/or C5 sugar, such as
compounds originating from a degradation process to liberate the C6
and C5 sugars from macromolecules. Such additional compounds may
e.g. be poly-, oligo- or disaccharides, furfural, salts etc., but
also lignin, and/or lignin-derived compounds and/or
compositions.
[0080] Non-limiting examples of C6 sugar are e.g. glucose,
galactose, mannose, rhamnose and the like. Non-limiting examples of
C5 sugar are xylose, arabinose etc. When a C6+C5 product is
obtained by hydrolysis of lignocellulosic material the C6 sugar
glucose is primarily obtained from the cellulose part whereas C5
sugar, mannose, galactose and rhamnose are primarily obtained from
the hemicellulose part of the lignocellulosic material. Said C5
and/or C6 sugar(s) may be modified, such as esterified or the
like.
[0081] In some embodiments, the C6 sugar is fermentable C6 sugar,
e.g. carbohydrates having six carbon atoms and which can be
fermented by well-known microorganisms, such as naturally occurring
microorganisms or genetically modified microorganisms.
[0082] In some embodiments, the C5 sugar is fermentable C5 sugar,
e.g. carbohydrates having five carbon atoms and which can be
fermented by well-known microorganisms such as naturally occurring
microorganisms or genetically modified microorganisms.
[0083] The term "C1-C4 product" as used herein means a small
molecular weight organic compound having from one to four carbon
atoms. Non-limiting examples of C1-C4 products are methanol,
ethanol, butanol, acetone, formic acid, acetic acid, propionic
acid, butyric acid, oxalic acid, lactic acid, malic aid, and/or any
combination thereof.
[0084] The term "Fermentation product" may comprise a C1-C4
product, as in the context of the current invention, the term
"fermentation product(s)" is meant to comprise any product that can
be provided by fermentation with one or more microorganism(s).
Fermentations according to the invention may comprise aerobic or
anaerobic fermentations, e.g. fermentations when pyruvate is
reduced to fermentation products such as ethanol, lactic acid, 3
hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propane-diol, ethylene, glycerol, butanol, a P-lactam
antibiotic and a cephalosporin. A "Fermentation product" may also
comprise value-added products including, but not limited to one or
more of: biofuels (including methanol, ethanol, propanol and
butanol); alcohol, aldehyde, ketone, lactic acid;
3-hydroxy-propionic acid; acrylic acid; acetic acid;
1,3-propane-diol; ethylene; glycerol; a plastic; a specialty
chemical; an organic acid, including citric acid, succinic acid and
maleic acid; a solvent; an animal feed supplement; a pharmaceutical
such as a p-lactam antibiotic or a cephalosporin; a vitamin; an
amino acid, such as lysine, methionine, tryptophan, threonine, and
aspartic acid; a peptide, a protein, an enzyme, such as a protease,
a cellulase, a hemicellulase, a xylanase, an amylase, a glucanase,
a lactase, a lipase, a lyase, an oxidoreductase, an esterase, or a
transferase; a chemical feedstock; or an animal feed
supplement.
[0085] "About" as used herein, usually with reference to a
quantitative number or range, may refer to +/- 1, 2, 5 or even 10%
in relative terms of the number or range referred to.
[0086] "Autohydrolysis" refers to a pretreatment process of
lignocellulosic biomass, in which acetic acid is liberated from
hemicellulose during said process, which is believed to further
catalyse and/or improve hemicellulose hydrolysis. Autohydrolysis of
lignocellulosic biomass is thus conducted without or essentially
without addition of any further chemicals, such as acid(s) or
base(s), and is commonly performed at a pH between 3.5 and 9.0.
[0087] "Commercially available cellulase preparation optimized for
lignocellulosic biomass conversion" refers to a commercially
available mixture of enzyme activities which is sufficient to
provide enzymatic hydrolysis of pretreated lignocellulosic biomass
and which usually comprises endocellulase (endoglucanase),
exocellulase (exoglucanase), endoxylanase, acetyl xylan esterase,
xylosidase and .beta.-glucosidase activities. The term "optimized
for lignocellulosic biomass conversion" refers to a product
development process in which enzyme mixtures have been selected
and/or modified for the specific purpose of improving hydrolysis
yields and/or reducing enzyme consumption in hydrolysis of
pretreated lignocellulosic biomass to fermentable sugars.
[0088] The term "Cellulase(s)" is meant to comprise one or more
enzymes capable of degrading cellulose and/or related compounds.
Cellulase is any of several enzymes commonly produced by fungi,
bacteria, and protozoans that catalyse cellulolysis, the
decomposition of cellulose and/or related polysaccharides.
Cellulase can also be used for any mixture or complex of various
such enzymes, that act serially or synergistically to decompose
cellulosic material. Cellulases break down the cellulose molecule
into monosaccharides ("simple sugars") such as beta-glucose, and/or
shorter polysaccharides and oligosaccharides. Specific reactions
may comprise hydrolysis of the 1,4-beta-D-glycosidic linkages in
cellulose, hemicellulose, lichenin, and cereal beta-D-glucans.
Several different kinds of cellulases are known, which differ
structurally and mechanistically. Synonyms, derivatives, and/or
specific enzymes associated with the name "cellulase" comprise
endo-1,4-beta-D-glucanase (beta-1,4-glucanase, beta-1,4-endoglucan
hydrolase, endoglucanase D, 1,4-(1,3,1,4)-beta-D-glucan
4-glucanohydrolase), carboxymethyl cellulase (CMCase), avicelase,
celludextrinase, cellulase A, cellulosin AP, alkali cellulase,
cellulase A 3, 9.5 cellulase, and pancellase SS.
[0089] Cellulases can also be classified based on the type of
reaction catalysed, where endocellulases (EC 3.2.1.4) randomly
cleave internal bonds at amorphous sites that create new chain
ends, exocellulases or cellobiohydrolases (EC 3.2.1.91) cleave two
to four units from the ends of the exposed chains produced by
endocellulase, resulting in tetra-, tri-or disaccharides, such as
cellobiose. Exocellulases are further classified into type I--that
work processively from the reducing end of the cellulose chain, and
type II--that work processively from the nonreducing end.
Cellobiases (EC 3.2.1.21) or beta-glucosidases hydrolyse the
exocellulase product into individual monosaccharides. Oxidative
cellulases depolymerize cellulose by radical reactions, as for
instance cellobiose dehydrogenase (acceptor). Cellulose
phosphorylases depolymerize cellulose using phosphates instead of
water.
[0090] The term "Hemicellulase(s)" is meant to comprise one or more
enzymes capable and/or contributing to breaking down hemicellulose,
one of the major components of plant cell walls. Some of the main
polysaccharides that constitute hemicellulose are believed to be
xylan, arabinoxylan, xyloglucan, glucuronoxylan and glucomannan. In
the context of the present invention, the term "hemicellulase(s)"
is meant to comprise: xylanase(s), xylosidase(s),
arabinoxylanase(s), xyloglucanase(s), glucoronoxylanase(s),
glucomannanase(s), and/or esterase(s), including any combination
thereof.
[0091] The term "Xylanase(s)" is meant to comprise one or more
enzymes capable of degrading xylan and/or related compounds.
Xylanase is any of several enzymes produced e.g. by microorganisms
such as yeast that catalyse decomposition of xylan and/or related
polysaccharides. Xylanase can also be used for any mixture or
complex of various such enzymes that act serially or
synergistically to decompose xylanosic material. Synonyms,
derivatives, and specific enzymes associated with the name
"xylanase" may comprise EC 3.2.1.8, endo-(1->4)-beta-xylan
4-xylanohydrolase, endo-1,4-xylanase, endo-1,4-beta-xylanase,
beta-1,4-xylanase, endo-1,4-beta-D-xylanase, 1,4-beta-xylan
xylanohydrolase, beta-xylanase, beta-1,4-xylan xylanohydrolase,
beta-D-xylanase and/or xylosidase capable of degrading xylan, such
as beta-1,4-xylan into xylose, thus contributing to breaking down
hemicellulose, one of the major components of plant cell walls.
[0092] "Xylosidase" as used herein is intended to comprise the
enzyme xylan 1,4-beta-xylosidase (E.C. 3.2.1.37) which is also
named xylobiase, beta-xylosidase, exo-1,4-beta-D-xylosidase or
4-beta-D-xylan xylohydrolase. This enzyme catalyses the hydrolysis
of (1-4)-beta-D-xylans removing successive D-xylose residues from
the non-reducing termini of the substrate, e.g. hemicellulose and
the disaccharide xylobiose. This enzyme is believed to be
commercially available both as an essentially pure xylosidase
enzyme or e.g. as a part of cellulase preparations.
[0093] The term "Arabinoxylanase(s)" is meant to comprise one or
more enzymes capable of degrading arabinoxylan and/or related
compounds, comprising e.g. glucuronoarabinoxylan
endo-1,4-beta-xylanase (EC 3.2.1.136), feraxan endoxylanase,
feraxanase, endoarabinoxylanase, glucuronoxylan xylohydrolase,
glucuronoxylanase, glucuronoxylan xylanohydrolase,
glucuronoarabinoxylan 1,4-beta-D-xylanohydrolase), and
glucuronoarabinoxylan 4-beta-D-xylanohydrolase.
Glucurono-arabinoxylan 4-beta-D-xylanohydrolase is believed to
endohydrolyse (1->4)-beta-D-xylosyl links in some
glucuronoarabinoxylans. It also believed that this enzyme possesses
a high activity towards feruloylated arabinoxylans. (Nishitani, K.;
Nevins, D. J. (1988). "Enzymic analysis of feruloylated
arabinoxylans (Feraxan) derived from Zea mays cell walls. I.
Purification of novel enzymes capable of dissociating Feraxan
fragments from Zea mays coleoptile cell wall". Plant Physiol. 87:
883-890.)
[0094] The term "Xyloglucanase(s)" is meant to comprise one or more
enzymes capable of degrading xyloglucan and/or related compounds,
comprising e.g. xyloglucan-specific endo-beta-1,4-glucanase (EC
3.2.1.151), which is an enzyme that is believed to catalyse the
chemical reaction: xyloglucan+H2O.fwdarw.xyloglucan
oligosaccharides. This enzyme belongs to the family of hydrolases,
specifically those glycosidases that hydrolyse O- and S-glycosyl
compounds. The systematic name of this enzyme class is
[(1->6)-alpha-D-xylo]-(1->4)-beta-D-glucan glucanohydrolase.
Other names in common use may include XEG, xyloglucan
endo-beta-1,4-glucanase, xyloglucanase, xyloglucanendohydrolase,
XH, and 1,4-beta-D-glucan glucanohydrolase.
[0095] The term "Glucuronoxylanase(s)" is meant to comprise one or
more enzymes capable of degrading glucuronoxylan and/or related
compounds.
[0096] The term "Glucomannanase(s)" is meant to comprise one or
more enzymes capable of degrading glucomannanase and/or related
compounds.
[0097] The term "Esterase(s)" is meant to comprise one or more
enzymes capable of splitting an ester in an acid and an alcohol.
Examples of esterases comprise acetylesterases and feroyl
esterase.
[0098] The term "Acetylesterase(s)" is meant to comprise an enzyme
capable of splitting off acetyl groups. An acetylesterase (EC
3.1.1.6) is an enzyme that catalyses the chemical reaction: [0099]
acetic ester+H2O.fwdarw.alcohol+acetate. This enzyme belongs to the
family of hydrolases, specifically those acting on carboxylic ester
bonds. The systematic name of this enzyme class is acetic-ester
acetylhydrolase. Other names in common use include C-esterase (in
animal tissues), acetic ester hydrolase, chloroesterase,
p-nitrophenyl acetate esterase, and Citrus acetylesterase.
[0100] The terms "Feroyl esterase(s)" and "Feruloyl esterase(s)"
can be used interchangeably, and is/are meant to comprise an enzyme
that catalyses the chemical reaction feruloyl-(poly-, oligo-, or
mono-) polysaccharide+H2O.fwdarw.ferulic acid+(poly-, oligo-, or
mono-)saccharide. Feroyl esterase belongs to the family of
hydrolases, specifically those acting on carboxylic ester bonds.
The systematic name of this enzyme class is feruloyl esterase (EC
3.1.1.73); other names may include ferulic acid esterase (FAE),
hydroxycinnamoyl esterase, hemicellulase accessory enzyme, and
cinnamoyl ester hydrolase (cinnAE).
[0101] Suitable microbial enzymes, such as cellulases,
hemicellulase(s) including xylanases, and or esterases, can be
expressed in suitable hosts using methods known in the art. Such
enzymes are also commercially available, either in pure form or in
enzyme cocktails. Specific enzyme activities can be purified from
commercially available enzyme cocktails, again using methods known
in the art--see e.g. Sorensen et al. (2005) "Efficiencies of
designed enzyme combinations in releasing arabinose and xylose from
wheat arabinoxylan in an industrial fermentation residue" (Enzyme
and Microbial Technology 36 (2005) 773-784), where a Trichoderma
reesei beta-xylosidase is purified from Celluclast (Finizym), and
further commercial enzyme preparations are disclosed.
[0102] Conducting a treatment/process, such as a pretreatment "at"
a dry matter level refers to the dry matter content of the
feedstock at the start of said treatment. Likewise, conducting a
treatment/process, such as a pretreatment "at" a pH refers to the
pH of the aqueous content of the biomass at the start of said
treatment.
[0103] In the context of the present invention, the term "pH- and
temperature-adjusted" is meant to comprise pH and/or temperature
adjustments in order to allow an enzymatic hydrolysis and/or
fermentation to take place under suitable pH and/or temperature
conditions.
[0104] "Dry matter," also appearing as "DM", refers to total
solids, both soluble and insoluble, and effectively means
"non-water content." Dry matter content is measured by drying at
105.degree. C. until constant weight is achieved. "Fiber structure"
is maintained to the extent that the average size of fiber
fragments following pretreatment is >750 .mu.m.
[0105] "Hydrothermal pretreatment" or sometimes only "pretreatment"
commonly refers to the use of water, either as hot liquid, vapour
steam or pressurized steam comprising high temperature liquid or
steam or both, to "cook" biomass, at temperatures of 120.degree. C.
or higher, either with or without addition of acids or other
chemicals. In the context of the present invention, "hydrothermal
pretreatment" is meant to comprise methods, unit operations and/or
processes relating to softening lignocellulosic biomass by the use
of temperature and water, and usually, also, pressure, aiming at
providing a pretreated biomass suitable for enzymatic
digestion.
[0106] "Single-stage pressurized hydrothermal pretreatment" refers
to a pretreatment in which biomass is subject to pressurized
hydrothermal pretreatment in a single reactor configured to heat
biomass in a single pass and in which no further pressurized
hydrothermal pretreatment is applied following a solid/liquid
separation step to remove liquid fraction from feedstock subject to
pressurized hydrothermal pretreatment.
[0107] "Process" water refers to water of a quality suitable for
the intended use in an industrial process. Commonly, process water
is of lower quality than e.g. drinking water. Process water may
comprise water that is recycled from an industrial process, such as
a process according to the present invention. Process water may be
adjusted in terms of mineral/salt content, pH and the like.
[0108] "Solid/liquid separation" refers to an active mechanical
process, and/or unit operation(s), whereby liquid is separated from
solid by application of force through e.g. pressing,
centrifugation, sedimentation, decanting or the like. Commonly, a
solid/liquid (s/l) separation provides a liquid and solid
fraction.
[0109] "Solid fraction" and "Liquid fraction" refer to
fractionation of pretreated and/or hydrolysed biomass in
solid/liquid separation. The separated liquid is collectively
referred to as "liquid fraction." The residual fraction comprising
considerable insoluble solid content is referred to as "solid
fraction". A "solid fraction" will have a substantial dry matter
content and typically will also comprise a considerable residual of
"liquid fraction" thus having the form of a solid or a slurry.
[0110] "Lignocellulosic biomass" refers to plant biomass comprising
cellulose and lignin, and usually also hemicellulose.
[0111] "Soft lignocellulosic biomass" refers to plant biomass other
than wood, which comprises cellulose and lignin, and usually also
hemicellulose.
[0112] The term "lignin" is meant to comprise a complex phenolic
polymer, which forms an integral part of the secondary cell walls
of various plants. It is believed that lignin is one of the most
abundant organic polymers on earth, exceeded only by cellulose, and
constituting from 25 to 33% of the dry mass of wood and 20 to 25%
for annual crops. "Lignin" is also used for a lignin component
obtained in the biomass refining process, usually comprising
pretreatment. Thus, the term "lignin" in the present description
and in the appended claims refers to the polymer denoted as such
and being present in unprocessed lignocellulosic plant material, as
well as "lignin" that has been subject to various physical and/or
chemical treatments, usually imposing only minor changes of the
lignin polymer structure, such as maintaining its polymer
character. Examples for such physical and/or chemical treatments
comprise processes and methods for providing a C5/C6 product as
disclosed herein. "Lignin" may comprise significant amounts of
hemicellulose and cellulose and/or other sugars. Hence "lignin" as
used in the present description and in the appended claims may
refer to a lignin that has been subjected to slight structural
modifications and/or comprising some amount of chemical residues
originating from its mode of manufacture, or originating from
compounds native for the lignocellulosic material from which it is
isolated.
[0113] In the context of the present invention, the term
"inhibitor" is meant to comprise one or more components or
chemicals reducing (i) the effectiveness of process, such as a
chemical reaction, e.g. catalysed by a catalyst such as an enzyme;
(ii) growth of a microorganism; and/or (iii) reducing metabolism,
in particular product yield, such as reduction in product yield of
a fermentation product. "Fermentation inhibitors" are inhibitors of
type (ii) and/or (iii). At least three categories of fermentation
inhibitors are typically formed during autohydrolysis pretreatment:
(1) furans, primarily 2-furfural and 5 hydroxymethylfurfural
(5-HMF) which are degradation products from mono- or
oligo-saccharides; (2) monomeric phenols, which are degradation
products of the lignin structure; and (3) small organic acids,
primarily acetic acid, which originate from acetyl groups in
hemicellulose and lignin. Further details concerning inhibitors
found in pretreated biomass, and methods of their determination and
analysis can e.g. be found in Rasmussen (2016) "Carbohydrate
degradation mechanisms and compounds from pretreated biomass" PhD
Thesis, Technical University of Denmark.
[0114] "Theoretical yield" refers to the molar equivalent mass of
pure monomer sugars obtained from polymeric cellulose, or from
polymeric hemicellulose structures, in which constituent monomeric
sugars may also be esterified or otherwise substituted. "C5 monomer
yields" as a percentage of theoretical yield are determined as
follows: Prior to pretreatment, biomass feedstock is analysed for
carbohydrates using strong acid hydrolysis and an HPLC system in
which galactose and mannose co-elute with xylose. Examples of such
systems are REZEX.TM., Monossacharide H+ column from Phenomenex and
an AMINEX HPX 87C.TM. column from Biorad. During strong acid
hydrolysis, esters and acid-labile substitutions are removed.
Except as otherwise specified, the total quantity of
"Xylose"+Arabinose determined in the un-pretreated biomass is taken
as a 100% theoretical C5 monomer recovery, which can be termed
collectively "C5 monomer recovery". Monomer sugar determinations
are made using HPLC characterization based on standard curves with
purified external standards. Actual C5 monomer recovery is
determined by HPLC characterization of samples for direct
measurement of C5 monomers, which are then expressed as a percent
of theoretical yield. "Xylan number" refers to a characterization
of pretreated biomass determined as follows: Pretreated biomass is
subject to solid/liquid separation to provide a solid fraction at
about 30% total solids and a liquid fraction. This solid fraction
is then partially washed by mixing with 70.degree. C. water in the
ratio of total solids (DM) to water of 1:3 wt:wt. The solid
fraction washed in this manner is then pressed to about 30% total
solids. Alternatively, the pretreated biomass can be subjected to
solid/liquid separation to provide a solid fraction at about 50%
total solids and a liquid fraction. With both methods, about 25% of
the dissolved solids remain in the solid fraction with the
suspended solids. Xylan content of the solid fraction washed in
this manner can determined using e.g. the method of A. Sluiter, et
al., "Determination of structural carbohydrates and lignin in
biomass," US National Renewable Energy Laboratory (NREL) Laboratory
Analytical Procedure (LAP) with issue date Apr. 25, 2008, as
described in Technical Report NREL/TP-510-42618, revised April
2008, which is expressly incorporated by reference herein in
entirety. This measurement of xylan content as described will
include some contribution of soluble material from residual liquid
fraction that is not washed out of solid fraction under these
conditions. Accordingly, in the context of the present invention,
the term "xylan number(s)" relates to (pre)treatment severities and
relates to a composite measurement and/or values that reflect a
weighted combination of both residual xylan content remaining
within insoluble solids and also the concentration of soluble
xylose and xylo-oligomers within the liquid fraction. At lower Ro
severity, xylan numbers are higher. Thus, the highest xylan number
refers to the lowest pretreatment severity. Xylan numbers provide a
negative linear correlation with the conventional severity measure
log R.sub.0 even to low severity, where residual xylan content
within insoluble solids is above 10%. Generally, low, medium and
high pretreatment severities provide xylan numbers of >10%,
6-10%, and <6%, respectively.
[0115] In the context of the present invention, unless indicated
otherwise, "%" indicates % weight/weight (w/w).
[0116] In the context of the present invention, the terms "about",
"around", "approximately" or the symbol ".about." can be used
interchangeably, and are meant to comprise variations generally
accepted in the field, e.g. comprising analytical errors and the
like. Thus "about" may also indicate measuring uncertainty commonly
experienced in the art, which can be in the order of magnitude of
e.g. +/-1, 2, 5, 10, 20, or even 50 percent.
[0117] The term "comprising" is to be interpreted as specifying the
presence of the stated parts, steps, features, components, or the
like, but does not exclude the presence of one or more additional
parts, steps, features, components etc. For example, a composition
comprising a chemical compound may thus comprise additional
chemical compounds.
[0118] A "derivative" is a compound that is derived from a similar
compound by a chemical reaction.
[0119] An "isomer" is a molecule with the same molecular formula as
another molecule, but with a different chemical structure. That is,
isomers contain the same number of atoms of each element, but have
different arrangements of their atoms. Isomers do not necessarily
share similar properties, unless they also have the same functional
groups. There are two main forms of isomerism: structural isomerism
(or constitutional isomerism) and stereoisomerism (or spatial
isomerism).
[0120] A "structural analogue", also known as a chemical analogue
or simply an analogue, is a compound having a structure similar to
that of another one, but differing from it in respect of a certain
component.
[0121] It can differ in one or more atoms, functional groups, or
substructures, which are replaced with other atoms, groups, or
substructures. A structural analogue can be imagined to be formed,
at least theoretically, from the other compound.
[0122] In the context of the present invention, terms related to
"recovering", "isolating", "purifying" and "concentrating" may be
used interchangeable, and are meant to comprise processes and/or
unit operations aiming at providing a desired product, compound,
and the like, such as a fermentation product or lignin in a more
concentrated, less contaminated and/or purer form. Suitable
processes, operations and/or processes are believed to be well
known in the art.
[0123] Lignocellulosic biomass comprises crystalline cellulose
fibrils intercalated within a loosely organized matrix of
hemicellulose and sealed within an environment rich in hydrophobic
lignin. While cellulose itself comprises long, straight chain
polymers of D-glucose, hemicellulose is a heterogeneous mixture of
short, branched-chain carbohydrates including monomers of all the
5-carbon aldopentoses (C5 sugars) as well as some 6-carbon (C6)
sugars including glucose and mannose. Lignin is a highly
heterogeneous polymer, lacking any particular primary structure,
and comprising hydrophobic phenylpropanoid monomers. Suitable
lignocellulosic biomass typically comprises cellulose in amounts
between 20 and 50% of dry mass prior to pretreatment, lignin in
amounts between 10 and 40% of dry mass prior to pretreatment, and
hemicellulose in amounts between 15 and 40%.
[0124] In some embodiments, biomass feedstocks may be subject to
particle size reduction and/or other mechanical processing such as
grinding, chopping, milling, shredding, cutting or other processes
prior to hydrothermal pretreatment. Other mechanical treatments may
comprise cleaning/purification means, such as means for removing
non-biomass components or objects, such as stones, grabble, sand,
dust, and/or foreign objects such as metal or plastic objects and
the like.
[0125] In some embodiments, biomass feedstocks may be washed and/or
leached of valuable salts prior to pressurized pretreatment. In
some embodiments, feedstocks may be soaked prior to pressurized
pretreatment at temperatures up to 99.degree. C. Said washing
and/or leaching is usually conducted at around environmental
pressure.
[0126] In some embodiments, the feedstock is first soaked in an
aqueous solution prior to hydrothermal pretreatment. In some
embodiments, the feedstock is soaked in an acetic acid containing
liquid obtained from a subsequent step in the pretreatments, as
described in U.S. Pat. No. 8,123,864, which is hereby incorporated
by reference in entirety. It may be advantageous to conduct
treatment at the highest possible dry matter content, as described
in U.S. Ser. No. 12/935,587, which is hereby incorporated by
reference in entirety. Conducting pretreatment at high dry matter
avoids expenditure of process energy on heating of unnecessary
water. However, some water content is required to achieve optimal
sugar yields from enzymatic hydrolysis. Typically, it is
advantageous to pretreat biomass feedstocks at or close to their
inherent water holding capacity. This is the level of water content
that a given feedstock will attain after soaking in an excess of
water followed by pressing to the mechanical limits of an ordinary
commercial screw press (typically between 30 and 45% DM). In some
embodiments, hydrothermal pretreatment is conducted at a DM content
of at least 35%. It will be readily understood by one skilled in
the art that the DM content may decrease during hydrothermal
pretreatment as some water content is added during heating. In some
embodiments, feedstocks are pretreated at a DM content of at least
20%, or at least 25%, or at least 30%, or at least 40%, or at less
than 40%, or at less than 35%, or at less than 30%. Further
suitable DM contents may be described elsewhere herein.
[0127] In some embodiments, soaking/wetting with an aqueous
solution can serve to adjust pH prior to pretreatment to the range
of between 3.5 and 9.0, which is typically advantageous for
autohydrolysis. It will be readily understood that pH may change
during pretreatment, typically to more acidic levels as acetic acid
is liberated from solubilized hemicellulose. Further suitable pH
values may be disclosed elsewhere herein.
[0128] Xylan number is particularly useful as a measure of
pretreatment severity in that different pretreated biomass
feedstocks having equivalent xylan number exhibit equivalent C5
monomer recovery. In contrast, conventional R.sub.0 severity is
simply an empirical description of pretreatment conditions, which
does not provide a rational basis for comparisons between different
biomass feedstocks. For example, single-stage autohydrolysis to
severity log R.sub.0=3.75 provides pretreated sugar cane bagasse
and corn stover having a xylan number of between 6-7%, while with
typical wheat straw varieties, the resulting xylan number of
pretreated feedstock is about 10%.
[0129] It may be advantageous that biomass feedstocks be pretreated
to low severity wherein xylan number of the pretreated feedstock is
greater 10% or greater. This low severity level corresponds to a
process in which the total hemicellulose content of the feedstock
before pretreatment that is either solubilized or irretrievably
lost during pretreatment is minimized. At xylan number 10% and
higher, with typical strains of wheat straw, sugar cane bagasse,
sweet sorghum bagasse, corn stover, and empty fruit bunches (from
oil palm), at least 60% of the original C5 content of the feedstock
can be recovered after single-stage autohydrolysis pretreatment,
where both xylan in the solid fraction and also soluble xylose and
xylo-oligomers in the liquid fraction are accounted for. High final
C5 monomer yields of at least 55% theoretical, at least 60%, or at
least 65%, can be obtained without appreciable loss of C6 monomer
yields after enzymatic hydrolysis of feedstocks pretreated to very
low severity by single-stage autohydrolysis. At very low severity
levels, a large fraction of the feedstock's hemicellulose content
remains within the solid fraction after pretreatment, where it can
subsequently be hydrolysed to C5 monomers with high recovery using
enzymatic hydrolysis.
[0130] It should be noted that reports concerning "xylose recovery"
are often expressed in terms that may not be directly comparable to
the xylose recoveries reported here. For example, reported xylose
recoveries often refer only to xylose recovery from pretreated
biomass, not expressed as a percentage of the original
hemicellulose content of the feedstock prior to pretreatment.
[0131] Another startling feature of biomass that has been
pretreated by single-stage autohydrolysis to very low severity
levels is that the concentrations of pretreatment by-products that
serve as inhibitors of fermentative organisms are kept to very low
levels. Consequently, it is often possible to use hydrolysed
biomass obtained by methods of the invention directly in
fermentations, without requirement for any washing or other
detoxification step. As is well known in the art, autohydrolysis
hydrothermal pretreatment typically produces a variety of soluble
by-products which act as "fermentation inhibitors," in that these
inhibit growth and/or metabolism of fermentative organisms.
Different fermentation inhibitors are produced in different
amounts, depending on the properties of the lignocellulosic
feedstock and on the severity of pretreatment. At least three
categories of fermentation inhibitors are typically formed during
autohydrolysis pretreatment: (1) furans, primarily 2-furfural and 5
hydroxymethylfurfural (5-HMF) which are degradation products from
mono- or oligo-saccharides; (2) monomeric phenols, which are
degradation products of the lignin structure; and (3) small organic
acids, primarily acetic acid, which originate from acetyl groups in
hemicellulose and lignin. The mixture of different inhibitors is
believed to act synergistically to inhibit microorganisms such as
yeasts and E. coli.
[0132] In some embodiments, pretreated biomass is subjected to
flash evaporation using methods well known in the art, in order to
reduce levels of volatile inhibitors, most notably furfural. When
using autohydrolysis with typical strains of biomass feedstocks,
such as wheat straw, sweet sorghum bagasse, sugar cane bagasse,
corn stover, and empty fruit bunches, pretreated to xylan number
10% or higher, it is believed that acetic acid and furfural levels
are potentially inhibitory to fermentative organisms. Where biomass
feedstocks are pretreated at DM 35% or higher to xylan number 10%
or higher, and where solid fraction is subsequently hydrolysed
enzymatically at 25% or lower DM, with added water to adjust DM but
without washing steps, furfural levels in the hydrolysate can
typically be kept under 3 g/kg and acetic acid levels beneath 9
g/kg. These levels are typically acceptable for yeast fermentations
using specialized strains. During enzymatic hydrolysis, some
additional acetic acid may be released from degradation of
hemicellulose in the solid fraction. In some embodiments, it may be
advantageous to remove some acetic acid content from liquid
fraction and/or hydrolysed solid fraction using electrodialysis
and/or other methods known in the art.
[0133] Lignocellulosic biomass, such as soft lignocellulosic
biomass feedstocks, such as agricultural waste such as cereal
straw, e.g. wheat, barley, rye or sorghum straw, grass, leaves,
sugar cane bagasse, sweet sorghum bagasse, corn stover, and empty
fruit bunches etc. are pretreated, usually preceded by a cleaning
step, where e.g. sand, stones, foreign objects and the like are
removed, and/or after a by single-stage autohydrolysis to xylan
number 10% or higher typically comprise a small component of C6
monomers (1.times.), primarily glucose with some other sugars; a
larger component of soluble C6 oligomers (about 2.times.-7.times.);
a larger component of C5 monomers (about 490.times.-8.times.),
primarily xylose with some arabinose and other sugars; and a much
larger component of soluble xylo-oligomers (about
18.times.-30.times.) wherein "nx" refers to the number of sugar
units, i.e. 1.times.=monomer, 2.times.=dimer, and so forth. Soluble
xylo-oligomers typically include primarily xylohexose, xylopentose,
xylotetraose, xylotriose and xylobiose with some higher chain
oligomers. Xylo-oligomers can also be modified, such as
esterified.
[0134] Different feedstocks can be pretreated using single-stage
autohydrolysis to e.g. xylan number 10% or greater by a variety of
different combinations of reactor residence times and temperatures.
One skilled in the art will readily determine through routine
experimentation an appropriate pretreatment routine to apply with
any given feedstock, using any given reactor, and with any given
biomass reactor-loading and reactor-unloading system. Where
feedstocks are pretreated using a continuous reactor, loaded by
either a sluice-system or a screw-plug feeder, and unloaded by
either a "particle pump" sluice system or a hydrocyclone system,
very low severity of 10% or greater xylan number can e.g. be
achieved using typical strains of wheat straw or empty fruit
bunches by a temperature of 180.degree. C. and a reactor residence
time of 24 minutes. For typical biomass feedstocks, such as soft
lignocellulosic biomass from commonly used varieties of corn
stover, sugar cane bagasse, and sweet sorghum bagasse, it is
believed that low severities, such as xylan numbers >10% can be
achieved using a temperature of around 180.degree. C. and a reactor
residence time of around 12 minutes, or using a temperature of
around 175.degree. C. and a reactor residence time of around 17
minutes. It will be readily understood by one skilled in the art
that residence times and temperatures maybe adjusted to achieve
comparable levels of R.sub.0 severity. Following pretreatment,
pretreated biomass is separated into a solid fraction and a liquid
fraction by a solid/liquid separation step. It will be readily
understood that "solid fraction" and "liquid fraction" may be
further subdivided or processed. In some embodiments, biomass may
be removed from a pretreatment reactor concurrently with
solid/liquid separation. In some embodiments, pretreated biomass is
subject to a solid/liquid separation step after it has been
unloaded from the reactor, typically using a simple and --low cost
screw press system, to generate a solid fraction and a liquid
fraction. Cellulase enzyme activities are inhibited by liquid
fraction, most notably due to xylo-oligomer content but possibly
also due to phenol content and/or other compounds not yet
identified. It can be advantageous to achieve the highest
practicable levels of dry matter content in the solid fraction or,
alternatively, to remove the highest practicable amount of
dissolved solids from the solid fraction. In some embodiments,
solid/liquid separation achieves a solid fraction having a DM
content of at least 40%, at least 45%, at least 50% or at least
55%. Solid/liquid separation using ordinary screw press systems can
typically achieve DM levels as high as 50% in the solid fraction,
especially when the biomass feedstock has been pretreated and
processed in such manner that fiber structure is maintained.
[0135] In some embodiments, it may be advantageous to incur higher
plant capital expenses in order to achieve more effective
solid/liquid separation, for example, using a membrane filter press
system. In some embodiments, dissolved solids can be removed from a
solid fraction by serial washing and pressing or by displacement
washing techniques known in the pulp and paper art. In some
embodiments, either by solid/liquid separation directly, or by some
combination of washing and solid/liquid separation, the dissolved
solids content of the solid fraction is reduced by at least 50%, at
least 55%, at least 60%, at least 65%, at least 70% or at least
75%. Enzymatic hydrolysis of feedstocks pretreated to xylan number
10% or higher can typically be conducted with commercially
reasonable enzyme consumption, without requirement for specific
washing or de-toxification steps, where the solid fraction is
pressed to at least 40% DM, or where dissolved solids content of
the solid fraction is reduced by at least 50%.
[0136] In some embodiments, hydrothermal pretreatment is conducted
without supplemental oxygen as required for wet oxidation
pretreatments, or without addition of organic solvent as required
for organosolv pretreatment, or without use of microwave heating as
required for microwave pretreatments. In some embodiments,
hydrothermal pretreatment is conducted at temperatures of
140.degree. C. or higher, or at 150.degree. C. or higher, or at
160.degree. C. or higher, or between 160 and 200.degree. C., or
between 170 and 190.degree. C., or at 180.degree. C. or lower, or
at 170.degree. C. or lower. In some embodiments, some C5 content
may be removed by a soaking step prior to pressurized pretreatment.
In some embodiments, the single reactor may be configured to heat
biomass to a single target temperature. Alternatively, the single
reactor may be configured to affect a temperature gradient within
the reactor such that biomass is exposed, during a single passage,
to more than one temperature region. In some embodiments, it may be
advantageous to partially remove some solubilized biomass
components from within the pressurized reactor during the course of
pretreatment.
[0137] Suitable hydrothermal pretreatment reactors typically
include most pulping reactors known from the pulp and paper
industry. In some embodiments, hydrothermal pretreatment is
administered by steam within a reactor pressurized to 10 bar or
lower, or to 12 bar or lower, or to 4 bar or higher, or 8 bar or
higher, or between 8 and 18 bar, or between 18 and 20 bar. In some
embodiments, the pretreatment reactor is configured for a
continuous inflow of feedstock.
[0138] In some embodiments, wetted biomass is conveyed through the
reactor, under pressure, for a certain duration or "residence
time." Residence time is advantageously kept brief to facilitate
higher biomass throughput. However, the pretreatment severity
obtained is determined both by temperature and by residence time.
Temperature during hydrothermal pretreatment is advantageously kept
lower, not only because methods of the invention seek to obtain a
very low pretreatment severity, but also because lower temperatures
can be accomplished using lower steam pressures. To the extent that
pretreatment temperature can be at levels of 180.degree. C. or
lower, and accordingly, saturated steam pressures kept to 10 bar or
lower, lower tendency for corrosion is experienced and much lower
grade pressure fittings and steel compositions may be used, which
reduces plant capital costs. In some embodiments, the reactor is
configured to heat biomass to a single target temperature between
160 and 200.degree. C., or between 170 and 190.degree. C.
[0139] Residence times in some embodiments are less than 60, less
than 30, less than 20, less than 15, less than 14, less than 3,
less than 12, less than 10, less than 8, or less than 5 minutes.
Further embodiments relating to suitable residence times may be
disclosed elsewhere.
[0140] Biomass feedstocks, such as lignocellulosic biomass, may be
loaded from atmospheric pressure into a pressurized reactor by a
variety of means. In some embodiments, a sluice-type "particle
pump" system may be used to load biomass feedstocks, such as the
systems described in e.g. WO 2003/013714 or WO 2011/024145, both of
which being hereby incorporated by reference in entirety. In some
embodiments, it may be advantageous to load a pretreatment reactor
using a so-called "screw plug" feeder.
[0141] Pretreated biomass may be unloaded from a pressurized
reactor by a variety of means. In some embodiments, pretreated
biomass is unloaded in such manner as to preserve the fiber
structure of the material. Preserving the fiber structure of the
pretreated biomass is advantageous because this permits the solid
fraction of the pretreated material to be pressed during
solid/liquid separation to comparatively high dry matter levels
using ordinary screw press equipment, and thereby avoiding the
added expense and complexity of membrane filter press systems.
Fiber structure can be maintained by removing the feedstock from
the pressurized reactor in a manner that is non-explosive. In some
embodiments, non-explosive removal may be accomplished and fiber
structure thereby maintained using sluice-type systems, such as
those described earlier. In some embodiments, non-explosive removal
may be accomplished and fiber structure thereby maintained using a
hydrocyclone removal system, such as those described in WO
2009/147512, which are hereby incorporated by reference in
entirety.
[0142] In some embodiments, pretreated biomass can be removed from
a pressurized pretreatment reactor using "steam explosion," which
involves explosive release of the pretreated material.
Steam-exploded, pretreated biomass does not retain its fiber
structure and accordingly requires more elaborate solid/liquid
separation systems in order to achieve dry matter content
comparable to dry matter contents, which can be achieved using e.g.
conventional screw press systems with pretreated biomass that
retains its fiber structure.
[0143] As will be readily understood by one skilled in the art, the
composition of enzyme mixtures suitable for practicing methods of
the invention may vary within comparatively wide bounds. Suitable
enzyme preparations include commercially available xylanase
preparations and cellulase preparations optimized for
lignocellulosic biomass conversion. Selection and modification of
enzyme mixtures during optimization may include genetic engineering
techniques. Commercially available cellulase preparations optimized
for lignocellulosic biomass conversion are typically identified by
the manufacturer and/or purveyor as such. These are typically
distinct from commercially available cellulase preparations for
general use or optimized for use in production of animal feed,
food, textiles detergents or in the paper industry. In some
embodiments, a commercially available cellulase preparation
optimized for lignocellulosic biomass conversion is used, such as
one that is e.g. provided by GENENCOR.TM. (now DuPont), DSM or
NOVOZYMES.TM.. Usually, such compositions comprise cellulase(s)
and/or hemicellulase(s), such as one or more of exoglucanases,
endoglucanases, endoxylanases, xylosidases, acetyl xylan esterases
and beta glucosidases, including any combination thereof. Such
enzymes can e.g. be isolated from fermentations of genetically
modified Trichoderma reesei, such as, for example, the commercial
cellulase preparation sold under the trademark ACCELLERASE
TRIO.TM..
[0144] In some embodiments, a commercially available cellulase
preparation optimized for lignocellulosic biomass conversion is
used that is provided by NOVOZYMES.TM. and that comprises
exoglucanases, endoglucanases, endoxylanases, xylosidases, acetyl
xylan esterases and beta glucosidases, such as, for example, the
commercial cellulase preparations sold under either of the
trademarks Cellic.RTM. CTec2 or Cellic.RTM. CTec3.
[0145] It is believed that the specific enzyme activities present
in different commercially available cellulase preparation optimized
for lignocellulosic biomass conversion can be analysed in detail
using methods known in the art.
[0146] Three different cellulase preparations, Accellerase.RTM.
TRIO.TM. from DuPont (and/or GENENCOR) and Cellic.RTM. CTec2 and
Cellic.RTM. CTec3 from NOVOZYMES.TM., are believed to be effective
at enzyme dose levels within the manufacturers' suggested
range.
[0147] Suitable cellulase preparations optimized for
lignocellulosic biomass conversion usually comprise multiple enzyme
activities, including exoglucanase, endoglucanase, hemicellulases
(including xylanases) and .beta.-glucosidases. Enzyme preparations
can be expressed in different activities/units, such as
carboxymethycellulase (CMC U) units, acid birchwood xylanase units
(ABXU), and pNP-glucosidase units (pNPG U). For example,
ACCELLERASE TRIO.TM. comprises: endoglucanase activity: 2000-2600
CMC U/g, xylanase activity: >3000 ABX U/g, and beta-glucosidase
activity: >2000 pNPG U/g; wherein one CMC unit of activity
liberates 1 .mu.mol of reducing sugars (expressed as glucose
equivalents) in one minute at 50.degree. C. and pH 4.8; one ABX
unit is defined as the amount of enzyme required to generate 1
.mu.mol of xylose reducing sugar equivalents per minute at
50.degree. C.; and pH 5.3; and one pNPG unit denotes 1 .mu.mol of
nitro-phenol liberated from para-nitrophenyl-B-D-glucopyranoside
per minute at 50.degree. C. and pH 4.8.
[0148] Based on the available information in the public domain, it
is believed that a person skilled in the art is able to provide
enzyme preparations suitable for enzymatic hydrolyses according to
the present invention, in particular for any one of the enzymatic
hydrolysis steps disclosed herein, such as fiber hydrolysis, fiber
cake hydrolysis and MSH (mixed sugar hydrolysis).
[0149] The current invention appears well suited for industrial
applications, including large-scale industrial applications. In
some embodiments, methods of the invention are practiced using at
least about 100, 200, 500 kg biomass feedstock, or at least 1000
kg, or at least 5000 kg.
[0150] In a first aspect, the current invention pertains to a
method for providing a C5/C6 product from a lignocellulosic
material comprising the steps: [0151] a) Pretreatment of the
lignocellulosic material; [0152] b) Solid/liquid separation of the
pretreated lignocellulosic material from step (a) into a first
solid fraction and a first liquid fraction; [0153] c) Enzymatic
fiber hydrolysis of said first solid fraction from step (b) by use
of an enzyme composition capable of degrading lignocellulosic
material, thereby providing a C5/C6 fiber slurry comprising C5
and/or C6 sugars; [0154] d) Solid/liquid separation of the C5/C6
fiber slurry from step (c) into a second solid fraction and a
second liquid fraction; and optionally [0155] e) Combining said
first liquid fraction and said second liquid fraction for enzymatic
mixed sugar hydrolysis (MSH), whereby a MSH C5/C6 product is
provided.
[0156] In some embodiments, said method may also comprise a further
step (f): Enzymatic fiber cake hydrolysis of said second solid
fraction from step (d) to obtain a slurry C5/C6 product.
[0157] According to the present invention, suitable lignocellulosic
biomass may comprise soft lignocellulosic biomass such as wheat
straw, corn stover, corn cobs, empty fruit bunches, rice straw, oat
straw, barley straw, canola straw, rye straw, sorghum, sweet
sorghum, soybean stover, switch grass, Bermuda grass and other
grasses, bagasse, beet pulp, corn fiber, or any combinations
thereof. Lignocellulosic biomass may comprise other lignocellulosic
materials such as wood, wood chips, but also paper, newsprint,
cardboard, or other municipal or office wastes. Lignocellulosic
biomass may be used as a mixture of materials originating from
different feedstocks, it may be fresh, partially dried, fully dried
or any combination thereof. Commonly, the lignocellulosic biomass
is considered a waste product.
[0158] In some embodiments, the lignocellulosic material is soft
lignocellulosic biomass, e.g. agricultural waste such as one or
more of wheat straw, corn stover, corn cobs, empty fruit bunches,
rice straw, oat straw, barley straw, canola straw, rye straw,
sorghum, sweet sorghum, soybean stover, switch grass, Bermuda grass
and other grasses, bagasse, beet pulp, corn fiber, or any
combinations thereof. In some embodiments, the lignocellulosic
biomass may also be predominantly or entirely ensiled biomass, or
comprise ensiled biomass, such as at least 5, 10, 25, 50%, 75%,
90%, 95%, 99% or more ensiled biomass.
[0159] In some embodiments, the lignocellulosic material is not
soft lignocellulosic biomass. Examples of such non-soft
lignocellulosic biomass comprise e.g. wood, wood chips, bark,
branches, but also paper, newsprint, cardboard, or even municipal
waste, such as sorted or unsorted municipal waste, or office
wastes. In some embodiments, the lignocellulosic biomass may also
be predominantly or entirely non-soft lignocellulosic biomass, or
comprise non-soft lignocellulosic biomass, such as at least 5, 10,
25, 50% or more than 50% non-soft lignocellulosic biomass.
[0160] In some embodiments, the pretreatment is conducted at a dry
matter (DM) content in the range of 5-80%, such as 10-70%, such as
20-60%, or such as 30-50%, or at a DM content around 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or
at a DM content of more than 80%. In some other embodiments, the
pretreatment is conducted at a DM content of 5-10%, 10-20%, 20-30%,
30-40%, 40-50%, 50-60%, 60-70%, or even 70-80%. In some further
embodiments, the pretreatment is conducted at a DM content of or
around 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80% or at a DM content of more than 80%.
[0161] In some embodiments, pretreatment is conducted at low,
medium, or high severity. In some embodiments, pretreatment is
conducted at conditions providing a xylan number of >10%, 6-10%
or <6%. It is believed that relevant advantages according to the
invention can also be obtained at medium, or high pretreatment
severities. In some embodiments, the biomass feedstock is
pretreated at medium severity, such that the pretreated biomass is
characterized by having a xylan number of 6-10%. In some
embodiments, the biomass is pretreated to a xylan number of 6-7%,
7-8%, or 9-10%. In further embodiments, the biomass feedstock is
pretreated at high severity, such that the pretreated biomass is
characterized by having a xylan number of less than 6%. In some
embodiments, the biomass is pretreated to a xylan number of below
6%, 5% or lower, 4% or lower, 3% or lower, 2% or lower, or 1% or
lower.
[0162] In some embodiments, enzymatic fiber hydrolysis, fiber cake
hydrolysis and/or MSH is/are conducted for a period of at least 6
h, 12 h, 24 h, 48 h, or 72 h, such as 6-120 h, 12-100 h, or 48-96
h, or around 12 h, 24 h, 48 h, 72 h, 96 h, or 120 h.
[0163] In some embodiments, enzymatic fiber hydrolysis, fiber cake
hydrolysis and/or MSH is/are conducted at a pH in the range of at
least pH 3.0, such as in the range of pH 3.0-6.0, such as pH
4.0-5.5, and/or such as pH 4.2-5.4.
[0164] In some embodiments, enzymatic fiber hydrolysis, fiber cake
hydrolysis and/or MSH is/are conducted at a pH of around 4.2, 4.5,
4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3 or 5.4.
[0165] In some embodiments, enzymatic fiber hydrolysis, fiber cake
hydrolysis and/or MSH is/are conducted at a temperature in the
range of 30-70.degree. C., 40-65.degree. C., 50-62.degree. C., or
55-60.degree. C., and/or around 40.degree. C., 42.degree. C.,
44.degree. C., 46.degree. C., 48.degree. C., 50.degree. C.,
52.degree. C., 54.degree. C., 56.degree. C., 58.degree. C.,
60.degree. C., 62.degree. C., 64.degree. C., 66.degree. C.,
68.degree. C., or 70.degree. C.
[0166] In some embodiments, enzymatic fiber hydrolysis, fiber cake
hydrolysis and/or MSH is/are conducted at a suitable DM content,
such as a DM content of at least 10%, such as 15%. In some
embodiments, the DM content is around 15-45%, 20-40%, 25-35%,
and/or at a DM content around 15%, 20%, 25%, 30%, 35%, or 40%. In
some embodiments, the DM content is around 40% or higher.
[0167] In some embodiments, the enzyme composition capable of
degrading lignocellulosic material comprises a cellulase and/or a
hemicellulase.
[0168] In some embodiments, the enzyme composition capable of
degrading lignocellulosic material comprises a mixture of
cellulase(s) and/or hemicellulase(s).
[0169] In some embodiments, the hemicellulase is or comprises one
or more xylanase(s), xylosidase(s), arabinoxylanase(s),
xyloglucanase(s), glucoronoxylanase(s), glucomannanase(s),
esterase(s), and any combination thereof.
[0170] Sugars, such as C5 and/or C6 mono-, oligo- and/or polymers
can be modified, such as esterified, e.g. comprising ferulic acid.
Ferulic acid can be efficiently released by esterase(s), such as a
ferulic acid esterase, e.g. FAE-III from Aspergillus niger (see
Faulds and Williamson, Appl. Microbiol. Biotechnol. 1995 November;
43(6): 1082-7), which released ferulic acid from wheat bran. Said
release was improved in the presence of a xylanase, such as a
Trichoderma viride xylanase. Hence, in some embodiments an
esterase, such as a ferulic acid esterase, and optionally cellulase
and/or at least one xylanase are added in an enzymatic hydrolysis
step, such as any one of fiber hydrolysis, fiber cake hydrolysis,
and/or mixed sugar hydrolysis.
[0171] In some embodiments, the esterases comprise one or more
acetylesterases and/or feroyl esterases.
[0172] In some embodiments, the enzyme composition capable of
degrading lignocellulosic material comprises one or more of
endocellulase(s), endoglucanase(s), exocellulase(s),
exoglucanase(s), endoxylanase(s), acetyl xylan esterase(s),
xylosidase(s), .beta.-glucosidase and any combination thereof.
[0173] In some embodiments, said method for providing a C5/C6
product from a lignocellulosic material comprises step (e), i.e.
combining said first liquid fraction and said second liquid
fraction for enzymatic mixed sugar hydrolysis (MSH). Through
combining said first liquid fraction and said second liquid
fraction and enzymatically hydrolysing the mixture, a MSH C5/C6
product is provided.
[0174] In some embodiments, hemicellulase(s) are also present in
step (e).
[0175] In some embodiments, the hemicellulase(s) present in step
(e) comprises xylanase(s), xylosidase(s), arabinoxylanase(s),
xyloglucanase(s), glucoronoxylanase(s), glucomannanase(s),
esterase(s), acetylesterases, feroyl esterase(s), and any
combination thereof.
[0176] In some embodiments, all or at least a fraction of the
hemicellulase(s) present in step (e) has been added in step
(c).
[0177] In some embodiments, one or more hemicellulase(s) is/are
added in step (e).
[0178] In some embodiments, wherein one or more additional
enzyme(s) are added in step (e). In some further embodiments, the
additional enzyme(s) is essentially not present (e.g. less than 1%
of total enzyme activity, present as only a minor side activity
and/or contamination etc.) in the enzyme composition capable of
degrading lignocellulosic material added in step (c). In other
embodiments, the additional enzyme(s) is one or more of:
hemicellulase(s), xylanase(s), xylosidase(s), arabinoxylanase(s),
xyloglucanase(s), glucoronoxylanase(s), glucomannanase(s),
esterase(s), acetylesterases, feroyl esterase(s), and any
combination thereof.
[0179] In some embodiments step e) comprises an ultrafiltration
step for separation of the hydrolysed sugars present in the
permeate from the hemicellulase(s) present in the retentate such as
to recycle at least part of the hemicellulase(s). Cost of enzymes
constitute a significant proportion of the variable costs of the
method, and the ultrafiltration step limits this cost by recycling
of the hemicellulase(s). Thus, in some embodiments, said step (e)
comprises or is followed by an ultrafiltration step (j) for
recycling enzymes present after MSH. In further embodiments, the
ultrafiltration step (j) is adapted to allow for recycling of at
least 30% (w/w), 50% (w/w), 75% (w/w), 80% (w/w), or 90% (w/w) of
the enzyme activity.
[0180] In some embodiments, cellulase(s) is/are also present in
step (f). In some embodiments, at least one cellulase has been
added such that said second solid fraction in step (f) comprises at
least one cellulase. In some embodiments, the cellulose(s) present
in step (f) has been added in step (c). Cellulases bind to the
fibers in the solid fraction and thus adding cellulase to the solid
fraction from step (b) may serve to complete the hydrolysis
performed in both step (c) (fiber hydrolysis) and step (f) (fiber
cake hydrolysis). Hence, in some embodiments, all, or essentially
all of the cellulase present in step (f) has been added in step
(c).
[0181] In some embodiments, at least a fraction of the cellulase
used in step (f) has been added in step (c).
[0182] In some embodiments, one or more cellulase(s) and optionally
hemicellulase(s) is/are added in step (f).
[0183] In some embodiments, cellulase(s) and/or hemicellulase(s)
are added in step (c), such as by the addition of a mixture
comprising one or more cellulase and one or more hemicellulase.
[0184] In some embodiments, the MSH and/or fiber cake hydrolysis
are performed without addition of one or more enzyme(s).
[0185] In some embodiments, the MSH and/or fiber cake hydrolysis
are performed without addition of one or more cellulose(s) and/or
one or more hemicellulose(s).
[0186] In some embodiments, said method for providing a C5/C6
product from a lignocellulosic material comprises step (g), namely
solid/liquid separation of the slurry C5/C6 product from step (f)
into a third solid fraction and a liquid C5/C6 product.
[0187] In some embodiments, the second liquid fraction possesses a
lower inhibitor concentration than the first liquid fraction.
[0188] In some embodiments, the second liquid fraction possesses a
lower inhibitor concentration than the MSH C5/C6 product.
[0189] In some embodiments, the slurry C5/C6 product" possesses a
lower inhibitor concentration than the MSH C5/C6 product.
[0190] In some embodiments, said method for providing a C5/C6
product from a lignocellulosic material comprises step (k), i.e.
combining at least a portion of the MSH C5/C6 product with at least
a portion of one or more of: the slurry C5/C6 product from step
(f), the liquid C5/C6 product from step (g), and/or the second
liquid fraction from step (d) to obtain a combined C5/C6
product.
[0191] In some embodiments, the combined C5/C6 product consists or
consists essentially of the MSH C5/C6 product and the slurry C5/C6
product from step (f); the MSH C5/C6 product and the liquid C5/C6
product from step (g); or the MSH C5/C6 product and the second
liquid fraction from step (d).
[0192] In some embodiments, the combined C5/C6 product possesses a
ratio of MSH C5/C6 product to liquid C5/C6 product; slurry C5/C6
product or second liquid fraction from step (d) in the range of
100:0.1-0.1:100 (w/w), such as 10:0.1-0.1:10 (w/w), or such as
10:1-1:10 (w/w), such as 5:1-1:5 (w/w); such as 4:1-1:4 (w/w), such
as 3:1-1:3 (w/w), such as 2.5-1:2.5 (w/w), such as 2:1-1:2 (w/w) or
such as 1.5-1:1-1.5 (w/w).
[0193] In some embodiments, the combined C5/C6 product possesses a
ratio of MSH C5/C6 product to liquid C5/C6 product; slurry C5/C6
product or second liquid fraction from step (d) is in the range
50:1 (w/w), 25:1 (w/w), 20:1 (w/w), 15:1 (w/w), 10:1 (w/w), 9:1
(w/w), 8:1 (w/w), 7:1 (w/w), 6:1 (w/w), 5:1 (w/w), 4:1 (w/w), 3:1
(w/w), 2:1(w/w), 1:1 (w/w), 1:1.5 (w/w), 1:2 (w/w), 1:2.5 (w/w),
1:3 (w/w), 1:4 (w/w), 1:5 (w/w), 1:6 (w/w), 1:7 (w/w), 1:8 (w/w),
1:9 (w/w), 1:10 (w/w), 1:15 (w/w), 1:20 (w/w), 1:25 (w/w), or 1:50
(w/w). In some preferred embodiments, said ratio is, or is about
1:1.5 (w/w), 1:2 (w/w), or 1:2.5 (w/w).
[0194] In some embodiments, said method for providing a C5/C6
product from a lignocellulosic material comprises a lignin recovery
step. This step may comprise on or more of: removal of water,
compacting and/or pelleting.
[0195] In some embodiments, said lignin recovery is conducted on
the second or third solid fraction provided in steps (d) or
(g).
[0196] In some embodiments, any C5/C6 product is a C5+C6 product,
i.e. a product comprising C5 and C6 carbohydrates, such as xylose
and glucose, including structural analogues, isomers and/or
derivatives thereof.
[0197] In some embodiments, the C5+C6 product comprises glucose and
xylose.
[0198] In a second aspect, the current invention relates to a
method for providing a fermentation product, said method comprising
the steps of: [0199] m) Providing at least one C5/C6 product
according to the method of any one of the preceding embodiments
according to the first aspect; and [0200] n) Providing the
fermentation product by a fermentation of said C5/C6 product with a
microorganism.
[0201] In some embodiments, the C5/C6 product comprises one or more
of: MSH C5/C6 product, Slurry C5/C6 product, Liquid C5/C6 product,
Combined C5/C6 product, first liquid fraction, or second liquid
fraction, and any combination thereof.
[0202] Usually, the fermentation product is provided in a
fermentation broth. Thus, in some embodiments said method of
providing a fermentation product comprising a further step (o):
recovering said fermentation product from a fermentation broth.
[0203] In some embodiments, said method comprises step (p):
recovering lignin from a spent fermentation broth, and/or a
fraction provided in steps (n) or (o).
[0204] In some embodiments, the fermentation is carried out in at
least a first and a second fermentation step, wherein a first and a
second fermentation substrate are fermented.
[0205] Providing two fermentation substrates with different
inhibitor and/or fermentation inhibitor concentration can be
advantageous, in particularly useful when the fermentation is
carried out by a microorganism sensitive to said inhibitors, which
are predominantly present in the C6+C5 product obtained in step b).
An increased productivity of the fermentation can thereby be
achieved, e.g. through a shorter duration of the fermentation, and
or higher product yield.
[0206] In particular, when also the combined fractions of the MSH
C5/C6 product+slurry or liquid C5/C6 product comprise too high
inhibitor concentration, the current invention provides an
alternative that does not require diluting the fermentation
substrate with water, which is not desirable. Such dilution with
water could be performed in a first fermentation, such as a batch
fermentation, before fermenting the combined fractions in e.g. a
fed-batch fermentation.
[0207] In some embodiments, the present invention relates to a
method as defined in the previous embodiments wherein said
fermentation is carried out by first batch-fermenting the liquid
C5/C6 product obtained e.g. in step (g) or the slurry C5/C6 product
obtained in step (f) and subsequently by fed-batch-fermenting the
MSH C5/C6 product obtained in step (e) or (j), usually in
combination with further quantities of liquid C5/C6 product
obtained e.g. in step (g) or the slurry C5/C6 product obtained in
step (f).
[0208] Further embodiments related to two-step fermentations are
also presented below.
[0209] In a third aspect, the current invention concerns a two-step
fermentation method comprising the steps of: [0210] aa)
Pretreatment of the lignocellulosic material; [0211] bb)
Solid/liquid separation of the pretreated lignocellulosic material
from step (aa) into a first solid fraction and a first liquid
fraction; [0212] cc) Enzymatic fiber hydrolysis of said first solid
fraction from step (bb) by use of an enzyme composition capable of
degrading lignocellulosic material, thereby providing a C5/C6 fiber
slurry; [0213] dd) Solid/liquid separation of the C5/C6 fiber
slurry from step (cc) into a second solid fraction and a second
liquid fraction; [0214] ee) Enzymatic mixed sugar hydrolysis (MSH)
of a mixture of the first liquid fraction from step (bb) and the
C5/C6 fiber slurry from step (cc), or the first liquid fraction
from step (bb) and the second liquid fraction from step (dd),
thereby providing a C5/C6 MSH product; [0215] ff) Providing a first
fermentation substrate comprising at least a portion of the C5/C6
fiber slurry and/or the second liquid fraction; [0216] gg)
Providing a second fermentation substrate comprising at least a
portion of the C5/C6 MSH product; [0217] hh) Fermenting the first
fermentation substrate in a first fermentation with a
microorganism; and [0218] ii) Fermenting the second fermentation
substrate in a subsequent second fermentation; [0219] wherein step
(dd) is optional.
[0220] In some embodiments, either one of steps aa, bb, cc, dd
and/or ee may correspond to steps a, b, c, d and/or e according to
any one of the previous aspects, respectively.
[0221] In some embodiments, the first fermentation substrate
possesses a significantly lower inhibitor concentration than the
second fermentation substrate.
[0222] In some embodiments, the first fermentation is a batch or
fed-batch fermentation.
[0223] In some embodiments, the first fermentation is carried out
by providing a first fermentation substrate comprising: (i) the
second liquid fraction provided in step (d) or (dd); (ii) the C5/C6
fiber slurry provided in step (c) or (cc); and/or (iii) the C5/C6
product obtained in step (f), i.e. the liquid C5/C6 product or the
slurry C5/C6 product.
[0224] In some embodiments, the first fermentation is carried out
by providing a first fermentation substrate consisting essentially
of: (i) the second liquid fraction provided in step (d) or (dd);
(ii) the C5/C6 fiber slurry provided in step (c) or (cc); and/or
(iii) the C5/C6 product obtained in step (f), i.e. the liquid C5/C6
product or the slurry C5/C6 product.
[0225] In some embodiments, the first fermentation substrate
comprises or consists essentially of a mixture of the second liquid
fraction and the C5/C6 product obtained in step (f), i.e. the
liquid C5/C6 product or the slurry C5/C6 product.
[0226] In some embodiments, the ratio between the second liquid
fraction and the C5/C6 product is in the range of 100:0.1-0.1:100
(w/w), such as 10:0.1-0.1:10 (w/w), or such as 10:1-1:10 (w/w).
[0227] In some embodiments, the ratio of the second liquid fraction
and the C5/C6 product is in the range of or around 50:1 (w/w), 25:1
(w/w), 20:1 (w/w), 15:1 (w/w), 10:1 (w/w), 9:1 (w/w), 8:1 (w/w),
7:1 (w/w), 6:1 (w/w), 5:1 (w/w), 4:1 (w/w), 3:1 (w/w), 2:1 (w/w),
1:1 (w/w), 1:2 (w/w), 1:3 (w/w), 1:4 (w/w), 1:5 (w/w), 1:6 (w/w),
1:7 (w/w), 1:8 (w/w), 1:9 (w/w), 1:10 (w/w), 1:15 (w/w), 1:20
(w/w), 1:25 (w/w), or 1:50 (w/w).
[0228] In some embodiments, the first fermentation substrate is
provided essentially without dilution with process water.
[0229] In some embodiments, the second fermentation is a fed-batch
fermentation or a continuous fermentation, optionally conducted in
the same fermenter as the first fermentation.
[0230] In some embodiments, said fed-batch fermentation is
conducted using linear or exponential feed.
[0231] In some embodiments, wherein the second fermentation is
conducted with the same microorganisms as in the first
fermentation.
[0232] In some embodiments, the second fermentation is carried out
by providing a second fermentation substrate comprising or
consisting essentially of a mixture of the C5/C6 product obtained
in step (f) (i.e. the liquid C5/C6 product or slurry C5/C6 product)
and the C5/C6 product obtained from step (e) (i.e. MSH C5/C6
product).
[0233] In some embodiments, the ratio between the liquid C5/C6
product or slurry C5/C6 product and the C5/C6 product obtained from
step (e) (i.e. MSH C5/C6 product) is in the range of
100:0.1-0.1:100 (w/w), such as 10:0.1-0.1:10 (w/w), or such as
10:1-1:10 (w/w), such as 5:1-1:5 (w/w); such as 4:1-1:4 (w/w), such
as 3:1-1:3 (w/w), such as 2.5-1:2.5 (w/w), such as 2:1-1:2 (w/w) or
such as 1.5-1:1-1.5 (w/w). In some preferred embodiments, said
ratio is 2.5-1:2.5 (w/w).
[0234] In some embodiments, the ratio between the liquid C5/C6
product or slurry C5/C6 product and the C5/C6 product obtained from
step (e) (i.e. MSH C5/C6 product) is in the range of or around 50:1
(w/w), 25:1 (w/w), 20:1 (w/w), 15:1 (w/w), 10:1 (w/w), 9:1 (w/w),
8:1 (w/w), 7:1 (w/w), 6:1 (w/w), 5:1 (w/w), 4:1 (w/w), 3:1 (w/w),
2:1 (w/w), 1:1 (w/w), 1:1.5 (w/w), 1:2 (w/w), 1:2.5 (w/w), 1:3
(w/w), 1:4 (w/w), 1:5 (w/w), 1:6 (w/w), 1:7 (w/w), 1:8 (w/w), 1:9
(w/w), 1:10 (w/w), 1:15 (w/w), 1:20 (w/w), 1:25 (w/w), or 1:50
(w/w). In some preferred embodiments, said ratio is, or is about
1:1.5 (w/w), 1:2 (w/w), or 1:2.5 (w/w).
[0235] In some embodiments, the second fermentation is carried out
by providing a second fermentation substrate comprising or
consisting essentially of the C5/C6 MSH product provided in step
(ee).
[0236] In some embodiments, the second fermentation is provided
essentially without dilution with of process water.
[0237] In some embodiments, the volume of the first fermentation is
significantly smaller than the volume of the second
fermentation.
[0238] In some embodiments, the volume of the first fermentation is
2-40%, 3-30%, 5-20%, 7.5-15%, 8-12%, or around 10% of the volume of
the second fermentation.
[0239] In some embodiments, the volume of the first fermentation is
around 5, 7.5, 10, 15, 20, 25, 30, 35 or 40% of the volume of the
second fermentation.
[0240] In some embodiments, the fermentation product is recovered
by distillation.
[0241] In some embodiments, said fermentation method comprises a
lignin recovery step, such as a lignin recovery step from a
distillation remnant.
[0242] In some embodiments, the first and second fermentation are
consecutive fermentations, optionally conducted in the same
fermenter.
[0243] In some embodiments, the second fermentation comprises
fermentation of both the first liquid fraction and the C5/C6 fiber
slurry.
[0244] In some embodiments, the fermentation product is an alcohol,
organic acid, vitamin, amino acid, peptide, enzyme or the like.
[0245] In some embodiments, the fermentation product is a C1-C4
product.
[0246] In some embodiments, the C1-C4 product is one or more of:
methanol, ethanol, butanol, acetone, formic acid, acetic acid,
propionic acid, butyric acid, oxalic acid, lactic acid, malic aid,
and/or any combination thereof.
[0247] In some embodiments, the C1-C4 product is EtOH.
[0248] In some embodiments, the microorganism is a eukaryotic or
prokaryotic microorganism, such as a bacterium or a yeast.
[0249] In some embodiments, the microorganism is a recombinant
microorganism.
[0250] In some embodiments, the microorganism is capable of
fermenting C5 and C6 sugars, such as xylose and glucose.
[0251] A variety of microorganisms may be used for the fermentation
of the C6+C5 product(s) into one or more fermentation product(s),
such as C1-C4 product such as ethanol, acetone and/or organic
acid(s), such as lactic or acetic acid, optionally also alone or in
combination with larger organic acids, such as valeric acid,
caproic acid, citric acid, or benzoic acid. As will be readily
understood by one skilled in the art, various yeast strains are
available which are suitable for converting C6 sugars as well as C5
sugars into ethanol, e.g. various Saccharomyces cerevisiae strains.
Also, for fermentations to produce lactic acid a range of suitable
microorganisms are available, such as lactic acid bacteria, such as
Lactococcus spp., Lactobacillus spp. etc. In some embodiments, the
microorganism is a Lactococcus spp., Lactobacillus spp.
[0252] In some embodiments, the microorganism is a yeast, such as a
Saccharomyces cerevisiae capable of or adapted to fermenting xylose
and glucose to EtOH.
[0253] In some embodiments, said fermentation is conducted by the
use of a microorganism, such as a recombinant microorganism capable
of converting C6 sugars and C5 sugars into ethanol.
[0254] In some embodiments, said fermentation is conducted by the
use of a recombinant microorganism capable of converting glucose
and xylose into ethanol.
[0255] In some embodiments, the fermentation is conducted with a
microorganism that is able to ferment at least one C5 sugar, apart
from one or more C6 sugar(s).
[0256] In some embodiment, the process is a process for the
production of ethanol, whereby the process comprises fermenting a
medium containing sugar(s) with a microorganism that is able to
ferment at least one C5 sugar, apart from one or more C6
sugar(s).
[0257] In some embodiments, the microorganism is able to ferment
glucose, L-arabinose and xylose to ethanol.
[0258] In some embodiments, the microorganism that is able to
ferment at least one C5 sugar, apart from one or more C6 sugar(s)
is a yeast. In an embodiment, the yeast belongs to the genus
Saccharomyces, preferably of the species Saccharomyces cerevisiae.
EP 1 499 708 describes a process for making S. cerevisiae strains
able to produce ethanol from L-arabinose.
[0259] WO2003/062430 and WO2006/009434 disclose yeast strains able
to convert xylose into ethanol. These yeast strains are able to
isomerise xylose into xylulose. In some embodiments, the
microorganism is a eukaryotic microorganism as disclosed in EP 1
499 708, WO2003/062430, WO2006/009434, or WO2008/041840.
[0260] In some embodiments, the microorganism is a genetically
modified yeast (e.g. Saccharomyces cerevisiae), capable of using
L-arabinose and/or to convert L-arabinose into L-ribulose, and/or
xylulose 5-phosphate and/or into a desired fermentation product.
Said microorganism may comprise the following genetic
modifications: (a) a cluster consisting of PPP-genes TAL1 TKL1,
RPE1 and RKI1, under control of strong promoters, (b) a cluster
consisting of a xyM-gene and the XKSi-gene both under control of
constitutive promoters, (c) a cluster consisting of the genes araA,
araB and araD and/or a cluster of xylA-gene and the XKSi-gene;
and/or (d) deletion of an aldose reductase gene.
[0261] In an embodiment, the fermentation process is anaerobic. In
another embodiment, the fermentation process is aerobic, optionally
under oxygen-limited conditions.
[0262] In an embodiment, the fermentation process is under
oxygen-limited conditions, such as a process in which the oxygen
consumption is limited by the oxygen transfer from the gas to the
liquid. The degree of oxygen limitation is determined by the amount
and composition of the ingoing gasflow as well as the actual
mixing/mass transfer properties of the fermentation equipment used.
Preferably, in a process under oxygen-limited conditions, the rate
of oxygen consumption is at least 5.5, more preferably at least 6
and even more preferably at least 7 mmol/L/h.
[0263] In a fourth aspect, the current invention concerns a method
for preparing ethanol and optionally lignin from a lignocellulosic
material comprising the steps of: [0264] Providing at least one
C5/C6 product according to a method according to any one embodiment
of the preceding aspects; [0265] Fermentation of said at least one
C5/C6 product to convert sugars to ethanol in the fermentation
broth with a yeast; [0266] Isolation of an ethanol rich fraction
from the fermentation broth; and optionally [0267] Isolation of
lignin.
[0268] In some embodiments, the fermentation is conducted according
to a method according any one embodiments relating to the second or
third aspect.
[0269] In some embodiments, lignin is isolated from the spent
fermentation broth or from the remnants from the spent fermentation
broth after isolating the ethanol rich fraction.
[0270] In a fifth aspect, the current invention pertains to lignin
provided from lignocellulosic biomass, such as ligning obtained or
obtainable according to any one of the preceding aspects. It is
believed that lignin provided according to the current invention,
in particular provided by a "V2.x" process is different from lignin
known in the art, such as lignin provided according to the a "whole
slurry" or V2 process. The lignin obtained is a high-value product
provided that the pre-treatment is not based on addition of acids
but e.g. conducted in the absence of added acids as described
above.
[0271] In a sixth aspect, the current invention relates to a C5/C6
product provided according to any one of the preceding aspects.
[0272] In a seventh aspect, the current invention concerns a
fermentation substrate comprising a C5/C6 product provided by a
method according to any one of the preceding aspects.
[0273] In an eighth aspect, the current invention pertains to a
first or second fermentation substrate provided by a method
according to any one of the preceding aspects.
[0274] In a ninth aspect, the current invention relates to
compositions comprising lignin obtained or obtainable by a method
according to any of the previous aspects, including different uses
of said lignin-comprising compositions.
[0275] In some embodiments, the present invention relates to lignin
obtained from the method according to any of the previous aspects,
such as a solid fraction from a spent fermentation broth or from
the distillation remnants from a distillation of a spent
fermentation broth.
[0276] In some embodiments, a composition is provided comprising
0.1-99.9, or 1-90% (w/w) lignin. It is believed that said lignin
can be used in bitumen compositions, including asphalt
compositions, such as bitumen compositions disclosed in
WO2017/088892, said document herewith being incorporated in its
entirity.
[0277] In some embodiments, a bitumen composition is provided
comprising: [0278] a. 1-99.89% (w/w) bitumen; [0279] b. 0.1-50%
(w/w) lignin; [0280] c. 0.01-20% (w/w) plasticity modifying
agent(s); and [0281] d. 0-95% (w/w) further component(s).
[0282] In some embodiments, said plasticity modifying agent is one
or more plastomer, one or more thermoplastic elastomer, one or more
rubber, one or more viscosity modifier, and/or one or more reactive
polymer, including any combination thereof.
[0283] In some embodiments, said further component(s) is one or
more dispersing agent(s), surfactant(s), hydrotropic agent(s),
emulsifier(s), preserving agent(s), anti-foaming agent (s),
viscosity modifier(s), reactive polymer(s) and any combination
thereof; and/or one or more aggregate(s) and/or filler(s), such
natural, manufactured, recycled aggregates, including any
combination thereof.
[0284] Said lignin-comprising compositions can be used in a wide
variety of applications. In some embodiments, said
lignin-comprising compositions can be used e.g. in sealing work,
road work, paving work, providing a surface layer, providing a
sealing layer, providing a road and providing a pavement, providing
a top layer of a road.
[0285] In some embodiments, said lignin-comprising compositions can
be used e.g. in applications relating to (i) agriculture, (ii)
buildings and industrial paving, (iii) hydraulics and erosion
control, (iv) industrial, (v) paving, (vi) railways, and (vii)
recreation, such as ad (i) disinfectants, fence post coating,
mulches, mulching paper, paved barn floors, barnyards, feed
platforms, protecting tanks, vats, protection for concrete
structures, tree paints (protective); ad (ii): water and moisture
barriers (above and below ground), floor compositions, tiles,
coverings, insulating fabrics, papers, step treads, building
papers, caulking compounds, cement waterproofing compounds, glass
wool compositions, insulating fabrics, felts, papers, joint filler
compounds, laminated roofing shingles, liquid roof coatings,
plastic cements, shingles, acoustical blocks, compositions, felts,
bricks, damp-proofing coatings, compositions, insulating board,
fabrics, felts, paper, masonry coatings, plasterboards, putty,
soundproofing, stucco base, wallboard, air-drying paints,
varnishes, artificial timber, ebonised timber, insulating paints,
plumbing, pipes, treated awnings, canal linings, sealants; ad
(iii): catchment areas, basins, dam groutings, dam linings,
protection, dyke protection, ditch linings, drainage gutters,
structures, embankment protection, groynes, jetties, levee
protection, mattresses for levee and bank protection, membrane
linings, waterproofing, reservoir linings, revetments, sand dune
stabilisation, sewage lagoons, oxidation ponds, swimming pools,
waste ponds, water barriers, backed felts, ad (iv): conduit
insulation, lamination, insulating boards, paint compositions,
papers, pipe wrapping, insulating felts, panel boards, underseal,
battery boxes, carbons, electrical insulating compounds, papers,
tapes, wire coatings, junction box compound, moulded conduits,
black grease, buffing compounds, cable splicing compound,
embalming, etching compositions, extenders, explosives, lap cement,
plasticisers, preservatives, printing inks, well drilling fluid,
armoured bituminised fabrics, burlap impregnation, mildew
prevention, sawdust, cork, asphalt composition, acid-proof enamels,
mastics, varnishes, acid-resistant coatings, air-drying paints,
varnishes, anti-corrosive and anti-fouling paints, anti-oxidants
and solvents, base for solvent compositions, baking and
heat-resistant enamels, boat deck sealing compound, lacquers,
japans, marine enamels, blasting fuses, briquette binders, burial
vaults, casting moulds, clay articles, clay pigeons, expansion
joints, flowerpots, foundry cores, friction tape, gaskets, mirror
backing, rubber, moulded compositions, shoe fillers, soles; ad (v):
airport runways, taxiways, aprons, asphalt blocks, brick fillers,
bridge deck, surfacing, crack fillers, floors for buildings,
warehouses, garages, highways, roads, streets, shoulders, kerbs,
gutters, drainage ditches, parking lots, driveways, Portland cement
concrete underseal, roof-deck parking, pavements, footpaths, soil
stabilisation; ad (vi) ballast treatment, dust laying, paved
ballast, sub-ballast, paved crossings, freight yards, station
platforms; and ad (vii) dance pavilions, drive-in movies,
gymnasiums, sport arenas, playgrounds, school yards, race tracks,
running tracks, skating rinks, swimming and wading pools, tennis
courts, handball courts, synthetic playing fields and running track
surfaces.
Comparison of "Whole Slurry" and "C5 Bypass" ("V2") Methods with
the Current Invention ("V2.X" Alias "Two Step Hydrolysis and Mixed
Sugar Hydrolysis")
[0286] Process scheme (1) (FIG. 2) shows a relatively simple
process configuration, such as a "whole slurry" processes described
in WO2015/014364: [0287] 1) Biomass, such as soft lignocellulosic
biomass, is steam pretreated at low severity (xylan number>10%,
such as 10-20%). [0288] 2) The pretreated biomass is pH- and
temperature-adjusted before enzymatic hydrolysis preferably in a
single step hydrolysis process (hence the name whole slurry
hydrolysis). [0289] 3) After enzymatic hydrolysis, the whole slurry
hydrolysate is pH- and temperature-adjusted before fermentation
with a suitable microorganism. The whole slurry hydrolysate is the
single substrate for microbial fermentation, such as a yeast
fermentation providing e.g. EtOH.
[0290] Process scheme (2) (FIG. 3) shows a more complex process
comprising a "C5 bypass", such as processes described in WO
2014/019589: "Methods of processing lignocellulosic biomass using
single-stage autohydrolysis and enzymatic hydrolysis with C5
by-pass and post-hydrolysis": [0291] 1) Biomass, such as soft
lignocellulosic biomass, is steam pretreated at low severity (xylan
number>10%, such as 10-20%). [0292] 2) The pretreated biomass is
separated (solid/liquid separation process) into a fiber fraction
(A) and a liquid fraction (B), said liquid fraction (B) comprising
C5 sugars (hence the name "C5 by-pass"). [0293] 3) The fiber
fraction (A) is diluted to a suitable dry matter content (e.g.
15-40% dry matter (DM)), and pH- and temperature-adjusted before
enzymatic hydrolysis. [0294] 4) The C5-bypass (liquid fraction (B))
is added at some point to the hydrolysing or hydrolysed fiber
fraction. It is believed that e.g. hemicellulose-derived oligomers,
such as xylan oligomers from the C5 by-pass are degraded to
monomers by enzymes as added in the fiber hydrolysis. [0295] 5) The
final hydrolysate is pH- and temperature-adjusted before
fermentation with a suitable microorganism. The final hydrolysate
is the single substrate for microbial fermentation, such as a yeast
fermentation providing e.g. EtOH.
[0296] Process scheme (3) (FIG. 4) depicts examples of a process
according to the current invention (also termed "V2.X" (or "two
step hydrolysis and mixed sugar hydrolysis")): [0297] 1)
Lignocellulosic biomass, such as soft lignocellulosic biomass, is
steam pretreated at a low severity in a single- or multiple-step
pretreatment process; medium or high pretreatment severities
comprise other options according to the present invention. [0298]
2) The pretreated biomass is separated into a first fiber fraction
("solid fraction-1") and a first liquid fraction ("liquid
fraction-1"). [0299] 3) The fiber fraction (A) is adjusted/diluted
to a suitable dry matter content (e.g. 15-40% DM), and pH- and/or
temperature-adjusted before enzymatic fiber hydrolysis. [0300] 4)
The hydrolysed fiber fraction is separated in to a fiber fraction
("solid fraction-2") and liquid fraction ("liquid fraction-2").
[0301] 5) "Solid fraction-2" is adjusted/diluted to a suitable dry
matter content, and pH- and/or temperature-adjusted before
enzymatic fiber cake hydrolysis. [0302] 6) "Liquid fractions-1 and
-2" are combined, and pH- and/or temperature adjusted before
hydrolysed with or without addition of additional enzymes (mixed
sugar hydrolysis). [0303] 7) Optionally, at least a fraction of the
mixed sugar hydrolysate can be subjected to ultra-filtration,
aiming as recycling at least a fraction of the enzymes, and adding
the recycled enzymes to the mixed sugar hydrolysis. [0304] 8)
Optionally, the fiber cake hydrolysate can be subjected to a
further solid/liquid separation step, providing a third fiber
fraction ("fiber fraction-3") and a third liquid fraction ("liquid
fraction-3") [0305] 9) Optionally, the hydrolysates from fiber cake
hydrolysis and/or mixed hydrolysis are pH-and/or
temperature-adjusted before fermentation with a suitable
microorganism, such as a yeast, providing EtOH.
[0306] This process provides different hydrolysates with different
levels of inhibitors. Thus, there is the option to feed the
fermentation from two hydrolysates with different content of
inhibiting substances formed in pretreatment, in particular
starting a fermentation with the hydrolysate with the lowest
concentration of inhibitors.
[0307] Furthermore, suitable enzyme preparations, can be added
either as enzyme mixes or single enzyme activities at different
process steps, such as at (i) fiber hydrolysis, (ii) fiber cake
hydrolysis and/or (iii) mixed sugar hydrolysis (see e.g. FIG. 1 or
4). In some embodiments, addition of further enzymes in any one of
said steps (ii) and/or (iii) is optional--this may not clearly be
depicted in said figures.
[0308] "Adjusted/diluted to a suitable dry matter content" before
fiber- and/or fiber cake hydrolysis may comprise the addition of
water, such as process water.
[0309] If available, e.g. when in close vicinity or in combination
suitable processing facility providing "raw juice"--i.e. a
water-base liquid comprising fermentable sugars, such as a 1 G EtOH
processing facility, or a sugar or fruit juice producing
facility--said dilution may comprise such a "raw juice".
[0310] Advantages of such a combination, such as water savings
and/or increased fermentation yields are disclosed in e.g.
WO2015/120859, or PCT/EP2016/069775, both applications being
herewith incorporated by reference in their entirety.
[0311] In summary, and without wanting to be construed as limiting,
the present invention may provide, inter alia, one or more of the
following effects and/or advantages: [0312] 1. increased C5/C6
product yield [0313] 2. reduced enzyme consumption [0314] 3.
addition of enzymes where they are needed [0315] 4. water savings
[0316] 5. cost savings, [0317] 6. improved lignin quality [0318] 7.
increased yield of fermentation product, such as C1-C4 product,
such as EtOH [0319] 8. reduced need for water
NUMBERED EMBODIMENTS
[0320] Relevant aspects and embodiments of the current invention
may also be found in the following section, termed "numbered
embodiments".
[0321] 2. A method for providing a C5/C6 product from a
lignocellulosic material comprising the steps: [0322] a)
Pretreatment of the lignocellulosic material; [0323] b)
Solid/liquid separation of the pretreated lignocellulosic material
from step (a) into a first solid fraction and a first liquid
fraction; [0324] c) Enzymatic hydrolysis ("fiber hydrolysis") of
said first solid fraction from step (b) by use of an enzyme
composition capable of degrading lignocellulosic material, thereby
providing a "C5/C6 Fiber hydrolysis slurry" comprising C5 and/or C6
sugars; [0325] d) Solid/liquid separation of the "C5/C6 Fiber
hydrolysis slurry" from step (c) into a second solid fraction and a
second liquid fraction; and optionally [0326] e) Combining said
first liquid fraction and said second liquid fraction for enzymatic
hydrolysis ("Mixed sugar hydrolysis (MSH)"), whereby a "MSH C5/C6
product" is provided.
[0327] 3. The method according to embodiment 1, comprising step
(f): enzymatic hydrolysis ("fiber cake hydrolysis") of said second
solid fraction from step (d) to obtain a "slurry C5/C6
product".
[0328] 4. The method according to embodiment 1 or 2, wherein the
lignocellulosic material is soft lignocellulosic biomass.
[0329] 5. The method according to any one of the preceding
embodiments, wherein the pretreatment is conducted at a dry matter
(DM) content in the range of 5-80, 10-70, 20-60, 30-50%, and/or at
a DM content around 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80 or more than 80%.
[0330] 6. The method according to any one of the preceding
embodiments, wherein the pretreatment is conducted at low, medium,
or high severity; and/or at conditions providing a xylan number of
>10%, 6-10% or <6%.
[0331] 7. The method according to any one of the preceding
embodiments, wherein the enzymatic fiber hydrolysis, fiber cake
hydrolysis and/or MSH is/are conducted for a period of at least 6,
12, 24, 48, or 72 h, such as 6-120 h, 12-100 h, or 48-96 h, or
around 12, 24, 48, 72, 96, or 120 h.
[0332] 8. The method according to any one of the preceding
embodiments, wherein the enzymatic fiber hydrolysis, fiber cake
hydrolysis and/or MSH is/are conducted at a pH in the range of at
least pH 3.0, such as 3.0-6.0, 4.0-5.5, 4.2-5.4, and/or around 4.2,
4.5, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3 or 5.4.
[0333] 9. The method according to any one of the preceding
embodiments, wherein the enzymatic fiber hydrolysis, fiber cake
hydrolysis and/or MSH is/are conducted at a temperature in the
range of 30-70.degree. C., 40-65.degree. C., 50-62.degree. C.,
55-60.degree. C., and/or around 40, 42, 44, 46, 48, 50, 52, 54, 56,
68, 60, 62, 64, 66, 68, or 70.degree. C.
[0334] 10. The method according to any one of the preceding
embodiments, wherein the enzymatic fiber hydrolysis and/or fiber
cake hydrolysis are conducted at suitable DM content, such as a DM
content above 10 or 15%, such as around 15-45, 20-40%, 25-35%,
and/or around 15, 20, 25, 30, 35, or 40%.
[0335] 11. The method according to any one of the preceding
embodiments, wherein the enzyme composition capable of degrading
lignocellulosic material comprises a cellulase and/or a
hemicellulase.
[0336] 12. The method according to any one of the preceding
embodiments, wherein the enzyme composition capable of degrading
lignocellulosic material comprises a mixture of cellulase(s) and/or
hemicellulase(s).
[0337] 13. The method according to any one of the preceding
embodiments, wherein the hemicellulase is or comprises one or more
xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s),
glucoronoxylanase(s), glucomannanase(s), and/or esterase(s),
including any combination thereof.
[0338] 14. The method according to embodiment 12, wherein the
esterase(s) is or comprises one or more acetylesterases and/or
feroyl esterase.
[0339] 15. The method according to any one of the preceding
embodiments, wherein the enzyme composition capable of degrading
lignocellulosic material comprises one or more of endocellulase,
endoglucanase, exocellulase, exoglucanase, endoxylanase, acetyl
xylan esterase, xylosidase and/or .beta.-glucosidase
activities.
[0340] 16. The method according to any of the preceding
embodiments, wherein step (e) is conducted by combining said first
liquid fraction and said second liquid fraction and enzymatically
hydrolysing the mixture.
[0341] 17. The method according to any of the preceding
embodiments, wherein hemicellulase(s) present in step (e) comprises
xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s),
glucoronoxylanase(s), glucomannanase(s), esterase(s),
acetylesterases, and/or feroyl esterase(s), including any
combination thereof.
[0342] 18. The method according to any of the preceding
embodiments, wherein all or at least a fraction of the
hemicellulase(s) used in step (e) has been added in step (c).
[0343] 19. The method according to any of the preceding
embodiments, wherein at least a fraction of the hemicellulase(s)
used in step (e) has been added in step (c).
[0344] 20. The method according to any one of the preceding
embodiments, wherein one or more hemicellulase(s) is/are added in
step (e).
[0345] 21. The method according to any one of the preceding
embodiments, wherein one or more additional enzyme(s) is provided
in step (e).
[0346] 22. The method according to embodiment 20, wherein the
additional enzyme(s) is essentially not present in the enzyme
composition capable of degrading lignocellulosic material
used/provided in step (c).
[0347] 23. The method according to embodiment 20 or 21, wherein the
additional enzyme(s) is one or more of: hemicellulase(s),
xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s),
glucoronoxylanase(s), glucomannanase(s), esterase(s),
acetylesterases, and/or feroyl esterase(s), including any
combination thereof.
[0348] 24. The method according to any of the preceding
embodiments, wherein step (e) comprises an ultrafiltration step (j)
for recycling enzymes present after MSH.
[0349] 25. The method according to embodiment 23, wherein the
ultrafiltration step (j) is adapted to allow for recycling of at
least 30, 50, 75, 80, or 90% (w/w) of the enzyme activity.
[0350] 26. The method according to any of the preceding
embodiments, wherein all the cellulase used in step (f) has been
added in step (c).
[0351] 27. The method according to any of the preceding
embodiments, wherein at least a fraction of the cellulase used in
step (f) has been added in step (c).
[0352] 28. The method according to any one of the preceding
embodiments, wherein one or more cellulase(s) and optionally
hemicellulase(s) is/are added in step (f).
[0353] 29. The method according to any one of the preceding
embodiments, wherein the MSH and/or fiber cake hydrolysis are
performed without addition of one or more enzyme(s).
[0354] 30. The method according to any one of the preceding
embodiments, wherein the MSH and/or fiber cake hydrolysis are
performed without addition of one or more cellulase and/or one or
more hemicellulase.
[0355] 31. The method according to any one of the preceding
embodiments further comprising the step: [0356] g) Solid/liquid
separation of the "slurry C5/C6 product" from step (f) into a third
solid fraction and a third liquid fraction ("liquid C5/C6
product").
[0357] 32. The method according to any one of the preceding
embodiments, wherein the second liquid fraction possesses a lower
inhibitor concentration than the first liquid fraction.
[0358] 33. The method according to any one of the preceding
embodiments, wherein the second liquid fraction possesses a lower
inhibitor concentration than the "MSH C5/C6 product".
[0359] 34. The method according to any one of the preceding
embodiments, wherein the "slurry C5/C6 product" possesses a lower
inhibitor concentration than the "MSH C5/C6 product".
[0360] 35. The method according to any one of the preceding
embodiments, further comprising the step: [0361] K) Combining at
least a portion of the "MSH C5/C6" product with at least a portion
of one or more of: the "slurry C5/C6 product" from step (f), the
"liquid C5/C6 product" from step (g), and/or the second liquid
fraction from step (d) to obtain a "combined C5/C6 product".
[0362] 36. The method according to embodiment 34, wherein the
"combined C5/C6 product" consists or consists essentially of the
"MSH C5/C6" product and the "slurry C5/C6 product" from step (f);
the "MSH C5/C6" product and the "liquid C5/C6 product" from step
(g); or the "MSH C5/C6" product and the second liquid fraction from
step (d).
[0363] 37. The method according to any one of the preceding
embodiments, further comprising a lignin recovery step, such as a
removal of water, compacting and/or pelleting.
[0364] 38. The method according to embodiment 36, wherein said
lignin recovery is conducted on the second or third solid fraction
provided in steps (d) or (g).
[0365] 39. The method according to any one of the preceding
embodiments, wherein any C5/C6 product is a C5+C6 product, i.e. a
product comprising C5 and C6 carbohydrates, such as xylose and
glucose, including structural analogues, isomers and/or derivatives
thereof.
[0366] 40. The method according to any one of the preceding
embodiments, wherein the C5+C6 product comprises glucose and
xylose.
[0367] 41. A method for providing a fermentation product, said
method comprising the steps of: [0368] m) Providing at least one
C5/C6 product according to the method of any one of the preceding
embodiments; and [0369] n) Providing the fermentation product by a
fermentation of said C5/C6 product with a microorganism. 42. The
method according to embodiment 40, wherein the C5/C6 product is or
comprises one or more of: "MSH C5/C6 product", "Slurry C5/C6
product", "Liquid C5/C6 product", "Combined C5/C6 product, first
liquid fraction, or second liquid fraction, including any
combination thereof.
[0370] 43. The method according to embodiment 40 or 41 wherein the
fermentation product is provided in a fermentation broth, said
method further comprising the step: (o) recovering said
fermentation product from a fermentation broth.
[0371] 44. The method according to any one of embodiments 40-42,
further comprising the step: (p) recovering lignin from a spent
fermentation broth, and/or a fraction provided in step (n) or
(o).
[0372] 45. The method according to embodiment 43, wherein
fermentation is carried in at least two subsequent fermentation
steps ("first and a second fermentation"), wherein a first and a
second fermentation substrate are fermented.
[0373] 46. A two-step fermentation method comprising the steps of:
[0374] aa) Pretreatment of the lignocellulosic material; [0375] bb)
Solid/liquid separation of the pretreated lignocellulosic material
from step (a) into a first solid fraction and a first liquid
fraction; [0376] cc) Enzymatic hydrolysis ("fiber hydrolysis") of
said first solid fraction from step (b) by use of an enzyme
composition capable of degrading lignocellulosic material, thereby
providing a "C5/C6 Fiber hydrolysis slurry"; [0377] dd)
Solid/liquid separation of the "C5/C6 Fiber hydrolysis slurry" from
step (cc) into a second solid fraction and a "second liquid
fraction"; [0378] ee) Enzymatic hydrolysis ("mixed sugar
hydrolysis" MSH) of a mixture of the first liquid fraction from
step (bb) and the "C5/C6 fiber hydrolysis slurry" from step (cc),
or the first liquid fraction from step (bb) and the second liquid
fraction from step (dd), thereby providing a "C5/C6 MSH product";
[0379] ff) Providing a first fermentation substrate comprising at
least a portion of the "C5/C6 fiber hydrolysis slurry" and/or the
second liquid fraction; [0380] gg) Providing a second fermentation
substrate comprising at least a portion of the C5/C6 MSH product;
[0381] hh) Fermenting the first fermentation substrate in a first
fermentation with a microorganism; and [0382] ii) Fermenting the
second fermentation substrate in a subsequent second fermentation;
wherein step (dd) is optional.
[0383] 47. The method according to any one of embodiments 44 or 45,
wherein the first fermentation substrate possesses a significantly
lower inhibitor concentration than the second fermentation
substrate.
[0384] 48. The method according to any one of embodiments 44-46,
wherein the first fermentation is a batch or fed-batch
fermentation.
[0385] 49. The method according to any one of embodiments 44-47,
wherein the first fermentation is carried out by providing a first
fermentation substrate comprising: [0386] x. the second liquid
fraction provided in step (d) or (dd); [0387] y. "C5/C6 fiber
hydrolysis slurry" provided in step (c) or (cc); and/or [0388] z.
the C5/C6 product obtained in step (f), i.e. the "liquid C5/C6
product" or the "slurry C5/C6 product".
[0389] 50. The method according to any one of embodiments 44-48,
wherein the first fermentation is carried out by providing a first
fermentation substrate consisting essentially of: [0390] x. the
second liquid fraction provided in step (d) or (dd); [0391] y.
"C5/C6 fiber hydrolysis slurry" provided in step (c) or (cc);
and/or [0392] z. the C5/C6 product obtained in step (f), i.e. the
"liquid C5/C6 product" or the "slurry C5/C6 product".
[0393] 51. The method according to any one of embodiments 44-49,
wherein the first fermentation substrate comprises or consists
essentially of a mixture of the second liquid fraction and the
C5/C6 product obtained in step (f), i.e. the "liquid C5/C6 product"
or the "slurry C5/C6 product".
[0394] 52. The method according to embodiment 50, wherein the ratio
of the second liquid fraction and the C5/C6 product is in the range
of 100:0.1-0.1:100, 10:0.1-0.1:10, or 10:1-1:10 (w/w); or around
50:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1,
2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20,
1:25, 1:50 (w/w).
[0395] 53. The method according to any one of embodiment 44-51,
wherein the first fermentation substrate is provided essentially
without dilution with process water.
[0396] 54. The method according to any one of embodiments 44-52,
wherein the second fermentation is a fed-batch fermentation or a
continuous fermentation, optionally conducted in the same fermenter
as the first fermentation.
[0397] 55. The method according to any one of embodiments 44-53,
wherein the fed-batch fermentation is with linear or exponential
feed.
[0398] 56. The method according to any one of embodiments 44-54,
wherein the second fermentation is conducted with the same
microorganisms as in the first fermentation.
[0399] 57. The method according to any one of embodiments 44-55,
wherein the second fermentation is carried out by providing a
second fermentation substrate comprising or consisting essentially
of a mixture of the C5/C6 product obtained in step (f) (i.e. the
"liquid C5/C6 product" or "slurry C5/C6 product") and the C5/C6
product obtained from step (e) (i.e. "MSH C5/C6 product").
[0400] 58. The method according to embodiment 56, wherein the ratio
of the "liquid C5/C6 product" or "slurry C5/C6 product") and the
C5/C6 product obtained from step (e) (i.e. "MSH C5/C6 product") is
in the range of 100:0.1-0.1:100, 10:0.1-0.1:10, or 10:1-1:10,
5:1-1:5; 4:1-1:4, 3:1-1:3, 2.5-1:2.5, 2:1-1:2 or 1.5-1:1-1.5 (w/w);
or around 50:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,
4:1, 3:1, 2:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7,
1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:50 (w/w).
[0401] 59. The method according to any one of embodiments 44-57,
wherein the second fermentation is carried out by providing a
second fermentation substrate comprising or consisting essentially
of the C5/C6 MSH product provided in step (ee).
[0402] 60. The method according to any one of embodiments 44-58,
wherein the second fermentation is provided essentially without
dilution with of process water.
[0403] 61. The method according to any one of embodiments 44-59,
wherein the volume of the first fermentation is significantly
smaller than the volume of the second fermentation.
[0404] 62. The method according to embodiment 60, wherein the
volume of the first fermentation is 2-40%, 3-30%, 5-20%, 7.5-15%,
8-12%, or around 10% of the volume of the second fermentation.
[0405] 63. The method according to any one of embodiments 40-61,
wherein the fermentation product is recovered by distillation.
[0406] 64. The method according to any one of embodiments 40-62,
wherein the fermentation product is EtOH.
[0407] 65. The method according to embodiment 62 or 63, further
comprising a lignin recovery step from a distillation remnant.
[0408] 66. The method according to any one of embodiments 44-64,
wherein the first and second fermentation are consecutive
fermentations, optionally conducted in the same fermenter.
[0409] 67. The method according to any one of embodiments 44-65,
wherein the second fermentation comprises fermentation of both the
first liquid fraction and the "C5/C6 Fiber hydrolysis slurry".
[0410] 68. The method according to any one of embodiments 40-66,
wherein the fermentation product is an alcohol, organic acid,
vitamin, amino acid, peptide, enzyme or the like.
[0411] 69. The method according to any one of embodiments 40-67,
wherein the fermentation product is a C1-C4 product.
[0412] 70. The method according to embodiment 68, wherein the C1-C4
product is one or more of: methanol, ethanol, butanol, acetone,
formic acid, acetic acid, propionic acid, butyric acid, oxalic
acid, lactic acid, malic aid, and/or any combination thereof.
[0413] 71. The method according to embodiment 68 or 69, wherein the
C1-C4 product is EtOH.
[0414] 72. The method according to any one of embodiments 40-70,
wherein the microorganism is a eukaryotic or prokaryotic
microorganism, such as a bacterium or a yeast.
[0415] 73. The method according to any one of embodiments 40-71,
wherein the microorganism is a recombinant microorganism.
[0416] 74. The method according to any one of embodiments 40-72,
wherein microorganism is capable of fermenting C5 and C6 sugars,
such as xylose and glucose.
[0417] 75. The method according to any one of embodiments 40-73,
wherein microorganism is a yeast, such as a Saccharomyces
cerevisiae capable of or adapted to fermenting xylose and glucose
to EtOH.
[0418] 76. A method for preparing ethanol and optionally lignin
from a lignocellulosic material comprising the steps of: [0419]
Providing at least one C5/C6 product according to a method
according to any one of the preceding embodiments; [0420]
Fermentation of said at least one C5/C6 product to convert sugars
to ethanol in the fermentation broth with a yeast; [0421] Isolation
of an ethanol rich fraction from the fermentation broth; and
optionally [0422] Isolation of lignin.
[0423] 77. The method according to embodiment 75, wherein the
fermentation is conducted according to a method according to any
one of embodiments 40-74.
[0424] 78. The method according to embodiment 75 or 76, wherein the
lignin is isolated from the spent fermentation broth or from the
remnants from the spent fermentation broth after isolating the
ethanol rich fraction.
[0425] 79. Lignin provided from lignocellulosic biomass according
to any one of the preceding embodiments.
[0426] 80. A C5/C6 product provided according to any one of the
preceding embodiments.
[0427] 81. A fermentation substrate comprising a C5/C6 product
provided by a method according to any one of the preceding
embodiments.
[0428] 82. The first or second fermentation substrate provided by a
method according to any one of the preceding embodiments.
[0429] 83. Use of lignin according according to embodiment 78 in a
bitumen composition, such as asphalt.
[0430] 84. A composition comprising 0.1-99.9% (w/w) lignin
according to embodiment 78.
[0431] 85. A bitumen composition comprising: [0432] a. 1-99.89%
(w/w) bitumen; [0433] b. 0.1-50% (w/w) lignin according to
embodiment 78; [0434] c. 0.01-20% (w/w) plasticity modifying
agent(s); and [0435] d. 0-95% (w/w) further component(s).
[0436] 86. The bitumen composition according to embodiment 84,
wherein the plasticity modifying agent is one or more plastomer,
one or more thermoplastic elastomer, one or more rubber, one or
more viscosity modifier, and/or one or more reactive polymer,
including any combination thereof.
[0437] 87. The bitumen composition according to embodiment 84 or
85, wherein the further component(s) is one or more dispersing
agent(s), surfactant(s), hydrotropic agent(s), emulsifier(s),
preserving agent(s), anti-foaming agent (s), viscosity modifier(s),
reactive polymer(s) and any combination thereof; and/or one or more
aggregate(s) and/or filler(s), such natural, manufactured, recycled
aggregates, including any combination thereof.
[0438] 88. A composition comprising 0.1-99.9% (w/w) lignin
according to embodiment 78.
[0439] 89. Use of a composition according to any one of embodiments
83-87 in sealing work, road work, paving work, providing a surface
layer, providing a sealing layer, providing a road and providing a
pavement, providing a top layer of a road
[0440] 90. Use of a composition according to any one of embodiments
83-87 in applications relating to (i) agriculture, (ii) buildings
and industrial paving, (iii) hydraulics and erosion control, (iv)
industrial, (v) paving, (vi) railways, and (vii) recreation, such
as ad (i) disinfectants, fence post coating, mulches, mulching
paper, paved barn floors, barnyards, feed platforms, protecting
tanks, vats, protection for concrete structures, tree paints
(protective); ad (ii): water and moisture barriers (above and below
ground), floor compositions, tiles, coverings, insulating fabrics,
papers, step treads, building papers, caulking compounds, cement
waterproofing compounds, glass wool compositions, insulating
fabrics, felts, papers, joint filler compounds, laminated roofing
shingles, liquid roof coatings, plastic cements, shingles,
acoustical blocks, compositions, felts, bricks, damp-proofing
coatings, compositions, insulating board, fabrics, felts, paper,
masonry coatings, plasterboards, putty, soundproofing, stucco base,
wallboard, air-drying paints, varnishes, artificial timber,
ebonised timber, insulating paints, plumbing, pipes, treated
awnings, canal linings, sealants; ad (iii): catchment areas,
basins, dam groutings, dam linings, protection, dyke protection,
ditch linings, drainage gutters, structures, embankment protection,
groynes, jetties, levee protection, mattresses for levee and bank
protection, membrane linings, waterproofing, reservoir linings,
revetments, sand dune stabilisation, sewage lagoons, oxidation
ponds, swimming pools, waste ponds, water barriers, backed felts,
ad (iv): conduit insulation, lamination, insulating boards, paint
compositions, papers, pipe wrapping, insulating felts, panel
boards, underseal, battery boxes, carbons, electrical insulating
compounds, papers, tapes, wire coatings, junction box compound,
moulded conduits, black grease, buffing compounds, cable splicing
compound, embalming, etching compositions, extenders, explosives,
lap cement, plasticisers, preservatives, printing inks, well
drilling fluid, armoured bituminised fabrics, burlap impregnation,
mildew prevention, sawdust, cork, asphalt composition, acid-proof
enamels, mastics, varnishes, acid-resistant coatings, air-drying
paints, varnishes, anti-corrosive and anti-fouling paints,
anti-oxidants and solvents, base for solvent compositions, baking
and heat-resistant enamels, boat deck sealing compound, lacquers,
japans, marine enamels, blasting fuses, briquette binders, burial
vaults, casting moulds, clay articles, clay pigeons, expansion
joints, flowerpots, foundry cores, friction tape, gaskets, mirror
backing, rubber, moulded compositions, shoe fillers, soles; ad (v):
airport runways, taxiways, aprons, asphalt blocks, brick fillers,
bridge deck, surfacing, crack fillers, floors for buildings,
warehouses, garages, highways, roads, streets, shoulders, kerbs,
gutters, drainage ditches, parking lots, driveways, Portland cement
concrete underseal, roof-deck parking, pavements, footpaths, soil
stabilisation; ad (vi) ballast treatment, dust laying, paved
ballast, sub-ballast, paved crossings, freight yards, station
platforms; and ad (vii) dance pavilions, drive-in movies,
gymnasiums, sport arenas, playgrounds, school yards, race tracks,
running tracks, skating rinks, swimming and wading pools, tennis
courts, handball courts, synthetic playing fields and running track
surfaces.
EXAMPLES
General Methods and Materials Used in Examples
[0441] In this part, the general methods and materials used for the
examples presented in this application are described. If deviated
from the general methods and materials, this will be specified in
the example.
Pretreatment
[0442] Pretreatment was conducted in Inbicon's pilot plant, Sk.ae
butted.rb.ae butted.k, Denmark. Wheat straw (WS) was soaked in
water, pH>4.0, prior to pretreatment at approximately 40% dry
matter (DM). About 50 kg DM/h of biomass was pretreated at
temperatures from 180-200.degree. C. with a residence time of
approximately 18 minutes. The biomass was loaded into the reactor
using a sluice system (WO2010/058285) and the pretreated material
unloaded again using a sluice system. The pressure within the
pressurized pretreatment reactor corresponded to the pressure of
saturated steam at the temperature used. The pretreated biomass was
subject to solid/liquid separation using a screw press, producing a
liquid fraction ("C5 bypass", "first liquid fraction") and a solid
fraction ("first solid fraction") with a DM content of
approximately 60%. The pretreatment process is further described in
Petersen et al. (2009).
Analytical Measurement of Pretreatment Fractions
[0443] Raw feedstocks were analysed for carbohydrates according to
the methods described in Sluiter et al. (2005) and Sluiter et al.
(2008) using a Dionex Ultimate 3000 HPLC system equipped with a
Rezex Monossacharide H+ column from Phenomenex.
[0444] Samples of liquid fraction and solid fraction were collected
after three hours of continuous pretreatment and samples were
collected three times over three hours to ensure that a sample was
obtained from steady state pretreatment.
[0445] The solid fractions were analysed for carbohydrates
according to the methods described in Sluiter et al. (2008) with an
Ultimate 3000 HPLC system from Dionex equipped with a Rezex
Monossacharide H+ Monosaccharide column from Phenomenex.
[0446] The liquid fractions were analysed for carbohydrates and
degradation products according to the methods described in Sluiter
et al. (2006) with an Ultimate 3000 HPLC system from Dionex
equipped with a Rezex Monossacharide H+ Monosaccharide column from
Phenomenex.
[0447] The total solids content (TS), hereafter termed dry matter
was measured by drying, approximately 24 hours, until constant
weight at 105.degree. C. The suspended solids (SS) were analysed
with a method adapted from the methods described in Weiss et al.
(2009) by analysing TS of the sample and TS in a sample filtered
through a paper filter and calculating the SS amount.
[0448] Mass balances were set up as described in Petersen et al.
(2009) and cellulose and hemicellulose recoveries were
determined.
Hydrolysis
[0449] Hydrolysis experiments were conducted in Inbicon's pilot
plant, Sk.ae butted.rb.ae butted.k, Denmark in two scales. Fiber
hydrolysis experiments were conducted in 10 kg scale in a free fall
reactor as described in WO2006/056838. The reactor is designed to
conduct experiments with a suspended dry matter content above 20%.
The reactor consists of a horizontally placed drum divided into 6
chambers, each 24 cm wide and 50 cm in height. A horizontal
rotating shaft mounted with three paddles in each chamber is used
for mixing/agitation. A 1.1 kW motor is used as a drive and the
rotational speed is adjustable within the range of 2.5 and 16.5
rpm. The direction of rotation is programmed to shift every second
minute between clock and anti-clockwise. A water-heated jacket on
the outside of the chambers enables temperature control up to
80.degree. C.
[0450] Hydrolysis experiments are conducted by adding fiber
fraction corresponding to 2.2 kg of suspended solids to a chamber
and then adding water or liquid fraction until the desired
separation degree of dissolved solids between the fiber and liquid
fraction is obtained in order to simulate a full-scale process. The
temperature is adjusted to 50.degree. C. The pH is adjusted to the
optimal pH for the used enzyme by use of Ca(OH).sub.2 prior to
addition of enzymes. The enzymes are added. Stirring is conducted
at 6 rpm. After liquefaction, the experiments were transferred to
shake flasks. The experiments are sampled after 4 hours and every
24 hours by sampling and diluting ten-fold and analysing according
to Kristensen et al. (2009) with an Ultimate 3000 HPLC system from
Dionex equipped with a Rezex Monossacharide H+ column from
Phenomenex.
Separation in Between Fiber Hydrolysis and Fiber Cake
Hydrolysis
[0451] After hydrolysis, the slurry was separated into a second
fiber fraction, fiber fraction-2, and a second liquid fraction,
liquid fraction-2, by pressing in a filter chamber press using one
cassette with Tetex Mono V05-1001-SK025 polypropylene filter cloth
at 60 to 65.degree. C. for ten minutes at 5 bars feeding pressure
and 13 bars pressing pressure.
Materials
[0452] Materials used are listed below in Table 1.
Example 1--Comparison of Total Carbohydrate Conversion in the V2.X
Method and the V2 (C5 By-Pass) Method
[0453] An example of the V2.X process is shown in process scheme 3
(FIG. 4). The main hypothesis behind the formation of the V2.X
process is--without wanting to be bound to any theory--that
significant and probably major parts of cellulases will follow the
fibers and main parts of the hemicellulases will follow the liquid
phase. Cellulases will be `reused` in a second fiber hydrolysis
step and hemicellulases will be reused in the mixed sugar
hydrolysis for hydrolysis of xylo- and other
hemicellulose-oligomers found in the liquid fraction-1 and liquid
fraction-2. As an option, ultra-filtration (UF) can be used to
up-concentrate the enzymes in the mixed sugar hydrolysis and/or to
improve hydrolysis yield. The fermentation process takes advantage
of and is becoming more efficient when using the two hydrolysates
with different content of inhibiting substances in the optimal
way.
[0454] Differences between the V2.X process (e.g. process scheme 3)
and the V2 or the C5 by-pass process (process scheme 2, FIG. 3)
comprise introduction of a two-step hydrolysis and/or that mixed
sugar hydrolysis is conducted without fibers.
[0455] In an experimental study, the glucan and xylan conversions
in the V2.X method and the C5 by-pass method have been compared
(FIG. 5). The first 72 hours of fiber hydrolysis is the same for
the V2.X method and the C5 by-pass method. The first 72 hours of
fiber hydrolysis was conducted in 10 kg scale in a free fall mixer.
After 72 hours of fiber hydrolysis, the slurry was split into two
fractions, a fraction to be used for continuing with V2.X method
and another fraction to be used for continuing with the C5 by-pass
method.
Fiber Hydrolysis for the V2.X Method and the C5 By-Pass Method
[0456] The fiber fraction was added to the chambers of the
free-fall reactor and water was added to reach a suspended dry
matter content of 22 wt-% giving a total dry matter content of 25
wt-%. The pH was adjusted to 5.3 and the temperature to 50.degree.
C. The agitator in the free fall mixer was set to 6 rpm. Five
chambers were used to compare the V2.X method and the C5 by-pass
method.
[0457] Table 2 shows the enzyme dosages used. After 72 hours at
50.degree. C. and pH adjusted in the range from 4.8-5.3, the fiber
hydrolysis was stopped.
TABLE-US-00001 TABLE 2 Enzyme dosages and SS for fiber hydrolysis
experiments Enzyme dosage Suspended [g Cellic .RTM. CTec3/kg dry
matter Experiment glucan in FH] [wt % SS] 16-13-R6-2 50 22
16-13-R6-3 40 22 16-13-R6-4 75 22 16-13-R6-5 50 22 16-13-R6-6 40
22
C5 By-Pass Method
[0458] The fraction to continue with C5 by-pass method was
transferred to shake flasks, which were placed in a shaking
incubator for 24 hours at 50.degree. C. After 24 hours, the C5
bypass (see section "pretreatment" above) was added and the
hydrolysis was continued for 50 hours without addition of enzymes.
Table 3 shows the enzyme dosages and the suspended dry matter (wt %
SS) used.
TABLE-US-00002 TABLE 3 Enzyme dosages and suspended dry matter (SS)
for mixed sugar hydrolysis (MSH) with V2 or C5 by-pass method
Enzyme dosage Suspended [g Cellic .RTM. CTec3/kg dry matter
Experiment glucan in FH] [wt % SS] 16-13-R6-2-FE-12-3 50 17
16-13-R6-3-FE-12-4 40 17 16-13-R6-4-FE-12-5 75 17
16-13-R6-4-FE-12-6 75 17 16-13-R6-5-FE-12-7 50 17
16-13-R6-6-FE-12-8 40 17
V2.X Method
[0459] The slurry fraction that continued in the V2.X method was
pressed into a fiber cake and a filtrate as described above. The
fiber cakes were allocated to six shake flasks and re-suspended in
water. Fiber cake hydrolysis was conducted for 72 hours with enzyme
dosages and SS % as shown in Table 4. The filtrate fraction was
transferred to shake flasks and the C5 bypass was added to start
the mixed sugar hydrolysis, which had a retention time of 48 hours.
Table 5 shows the enzyme dosage and % SS in the MSH. Both the fiber
cake hydrolysis and the MSH were conducted at 50.degree. C. and pH
5.0-5.3. The agitation for the fiber cake hydrolysis and the MSH
were set to 250 rpm in the shaking incubator, see also FIG. 5 for
an overview of the setup.
TABLE-US-00003 TABLE 4 Enzyme dosages and SS for fiber cake
hydrolysis with V2.X method Enzyme dosage Suspended [g Cellic .RTM.
CTec3/kg dry matter Experiment glucan in FH] [wt % SS]
16-13-R6-2-FE-13-14 50 19 16-13-R6-3-FE-13-17 40 19
16-13-R6-4-FE-13-20 75 18 16-13-R6-5-FE-13-23 50 19
16-13-R6-6-FE-13-26 40 18
TABLE-US-00004 TABLE 5 Enzyme dosages and SS for MSH with V2.X
method Enzyme dosage Suspended [g Cellic .RTM. CTec3/kg dry matter
Experiment glucan in FH] [wt % SS] 16-13-R6-2-FE-13-28 50 0
16-13-R6-3-FE-13-29 40 0 16-13-R6-4-FE-13-30 75 0
16-13-R6-5-FE-13-31 50 0 16-13-R6-6-FE-13-32 40 0
Results
[0460] After all the hydrolysis were conducted, the sugar
concentrations were measured and mass balances were calculated. The
glucan, xylan and arabinan conversions were calculated as sum of
glucose, xylose and arabinose after mixed sugar hydrolysis and
fiber cake hydrolysis divided by the sum of glucan, xylan and
arabinan respectively in the fiber fraction and the C5-bypass. The
glucan conversion calculated based on total amount of glucan from
pretreatment increases with 11-17% (relatively), when using the
V2.X method compared to the C5 by-pass method, see FIG. 6. The
xylan conversion calculated based on total amount of xylan from
pretreatment is similar respectively for the V2.X method and the C5
by-pass method, which confirms that most of the xylanases follow
the filtrate after the press of the slurry from the fiber
hydrolysis (FIG. 7). The arabinan conversion calculated based on
total amount of arabinan from pretreatment increases with 7 to 19%
(relatively) when using the V2.X method compared to the C5 by-pass
method, which shows that other hemicellulases than xylanases follow
the filtrate (FIG. 8).
Conclusion
[0461] Hydrolysis experiments were conducted in 10 kg scale at
industrial relevant dry matter with three different enzyme dosages
to compare the V2 (C5 by-pass method) and the V2.X method. In all
experiments, better yields were obtained for overall monomeric
carbohydrate yield in the V2.X method. The average increase
observed was 8% more absolute conversion of glucan to glucose, no
significant change in conversion of xylan to xylose is observed and
7% more absolute conversion of arabinan to arabinose.
Example 2--Comparison of Carbohydrate Conversion of the Fiber
Fraction in the V2.X Method and the V2 (C5 By-Pass) Method with
Multiple Pretreatments and Biomasses
[0462] The V2.X process was tested with different wheat straw
batches and different pretreatments (see also Table 6). The
comparison between the V2.X method and the V2 or C5 by-pass method
was based on the enzymatic conversion of the fiber fractions. The
total enzyme dose to fiber fraction was similar; 75 g Cellic.RTM.
CTec3/kg total glucan in FH. The enzyme was added in one portion to
the fiber hydrolysis in the C5 by-pass method while added in two
steps distributed to fiber and fiber cake hydrolysis in the V2.X
method. In both cases, no mixed sugar hydrolysis was conducted.
Comparison of mixed sugar hydrolysis in both methods is described
in example 5.
Fiber Hydrolysis for the C5 By-Pass Method
[0463] The fiber fraction was added to the chambers of the
free-fall reactor and water added to reach a SS of 22 wt % giving a
total dry matter content of 25 wt %. The pH was adjusted to 5.3 and
the temperature to 50.degree. C. The agitator in the free-fall
mixer was set to 6 rpm. After approx. 100 hours at 50.degree. C.
and pH adjusted in the range from 4.8-5.3 the fiber hydrolysis was
stopped. In one case (16-13-R6-4), the fiber mash was removed from
the free-fall reactor after 72 h and fiber hydrolysis was continued
in shake flasks for another 24 h.
TABLE-US-00005 TABLE 6 Enzyme dosage for fiber hydrolysis
experiment with C5 by-pass method Wheat Enzyme dosage straw
Pretreatment Hydrolysis [g Cellic .RTM. CTec3/kg batch experiment
experiment glucan in FH] WS_F WS_F_20150729 15-71-R6-1 75 WS_F
WS_F_20150902 15-71-R6-2 75 WS_F WS_F_20150923 16-4-R6-4 75 WS_F
WS_F_20140828 16-4-R6-2 75 WS_H WS_H_20160203 16-13-R6-4 75
Fiber and Fiber Cake Hydrolysis for the V2.X Method
[0464] The fiber fraction was added to the chambers of the
free-fall reactor and water added to reach a SS of 22 wt-% giving a
total dry matter content of 25 wt-%. The pH was adjusted to 5.3 and
the temperature to 50.degree. C. The enzyme dosage for this
experiment is given in Table 7. The agitator in the free fall mixer
was set to 6 rpm. After approx. 72 hours at 50.degree. C. and pH
adjusted in the range from 4.8-5.3 the fiber hydrolysis was
stopped. The slurry was pressed into a fiber cake and filtrate as
previously described. The fiber cakes were allocated to five shake
flasks and re-suspended in water and the second portion of enzymes
was added. The fiber cake hydrolysis was conducted for 68 to 72
hours at 50.degree. C. and pH 5.0-5.3. The agitation for the fiber
cake hydrolysis was set to 250 rpm in the shaking incubator.
TABLE-US-00006 TABLE 7 Enzyme dosages for fiber and fiber cake
hydrolysis with V2.X method Enzyme dosage, Enzyme dosage, Wheat
Fiber Fiber Cake straw Pretreatment Hydrolysis Hydrolysis
Hydrolysis batch experiment experiment [g Cellic .RTM. CTec3/kg
glucan in FH] WS_F WS_F_20150729 15-78-R6-1-FE-48-1/2/11/12 50 25
WS_F WS_F_20150902 15-78-R6-2-FE-48-3/4 50 25 WS_F WS_F_20150923
15-78-R6-4-FE-48-5/6 50 25 WS_F WS_F_20140828 15-78-R6-5-FE-48-7/8
50 25 WS_H WS_H_20160203 16-13-R6-2/5-FE-13-4/13 50 25
Results
[0465] At the end of the hydrolysis, the sugar concentrations in
all streams were measured and mass balances were set up. The glucan
and xylan conversions of the fibers from pretreatment were
calculated based on monomeric sugar concentrations in the fiber
slurry (C5 by-pass method) or as sum of the filtrate and fiber cake
slurry. Glucan and xylan conversions are shown in FIGS. 9 and
10.
Conclusion
[0466] The hydrolysis yield of fibers after pretreatment has been
compared for two different wheat straw batches and five different
pretreatment dates. In all the trials a significantly better glucan
conversion, 13% (relatively) more in V2.X compared to V2, of the
fibers has been achieved by performing two stage hydrolysis (V2.X).
The xylan conversion showed in most cases also an improved
performance with two-stage hydrolysis (V2.X), giving a mean
increase of 8% (relatively) more xylose from V2.x compared to
V2.
[0467] The conclusion is that V2.X is yielding higher than the C5
by-pass (V2) method over a series of experiments with varied
biomass composition and repeated pretreatment experiments with
approx. 500 kg pretreated wheat straw processed in each
experiment.
Example 3--Comparison of Two-Stage Hydrolysis with Enzyme Dose
Split
[0468] In practical experiments it has been proven that two-stage
hydrolysis yields are higher than single stage hydrolysis yields,
but the effect is not high if all enzyme is added in the fiber
hydrolysis. A significant higher yield is obtained with two stage
hydrolysis, when the enzyme dose is split to both stages.
[0469] Three fiber hydrolyses were conducted in the free fall
reactor in 10 kg scale, one with 50 g CTec3/kg glucan and 22 wt %
SS and another two with standard 75 g CTec3/kg glucan; one with 22
wt-% SS and another with 18 wt % SS corresponding to the final dry
matter of a two-stage hydrolysis with 22 wt % SS in both fiber
hydrolysis and fiber cake hydrolysis. Otherwise standard hydrolysis
conditions.
[0470] After 44 hours of fiber hydrolysis, the slurries was pressed
into a filtrate and a fiber cake as previously described. The fiber
cake was re-suspended in water to 22% SS in shake flasks. For the
chamber with 50 g CTec3/kg glucan in the fiber hydrolysis, the rest
of the enzyme up to a total enzyme dose of 75 g CTec3/kg original
glucan was added to the fiber cake hydrolysis. No enzymes were
added to the fiber cake for the trial with 75 g CTec3/kg glucan in
the fiber hydrolysis.
Results
[0471] FIG. 11 shows the results of one and two stage hydrolysis
and dependence of dry matter. The lowest conversion (71%) is
obtained conducting one stage hydrolysis at 22% SS. The yield is
improved in a one stage hydrolysis to 74% if the SS in the
hydrolysis is lowered from 22 to 18 wt-% SS. The water consumption
in a 18 wt-% SS one stage hydrolysis equals the water consumption
in a 22 wt-% SS two-step hydrolysis (because of the two steps at
22% SS). Therefore it is not unexpected that the yield from a one
stage hydrolysis at 18% SS is yielding comparable conversion as a
two step hydrolysis at 22 wt-% SS, although a small increase in
yield is expected due to lower product inhibition in the fiber cake
hydrolysis. The yield increases from 74 to 76% going from a one
stage hydrolysis to a two stage hydrolysis when maintaining the
same water consumption in the overall process. A significant higher
yield is obtained with two stage hydrolysis, when the enzyme dose
is split and dosed in both stages. By adding only two thirds of the
enzyme to the fiber hydrolysis and the rest (one third) of the
enzyme to the fiber cake hydrolysis, the glucan conversion
increases from 76% to 82%, see FIG. 11.
Conclusion
[0472] Going from one to two stage hydrolysis increased the glucan
conversion by 6% (relatively) when maintaining the same water
consumption in the process. It is of advantage to add some of the
enzyme to the fiber hydrolysis and the rest of the enzyme to the
fiber cake hydrolysis. This way of enzyme dosing will enhance the
effect of two-step hydrolysis, giving an increase in glucan
conversion of 16% (relatively) rather than only 6%
(relatively).
Example 4--Comparison of Mixed Sugar Hydrolysis (MSH) with Fibers
(C5 Bypass Method) and Without Fibers (V2.X Method)
[0473] In the V2.X process, process scheme (3), the MSH is a
mixture of sugar juice from the fiber hydrolysis and liquid
fraction-1. MSH is mixed in a volume ratio of approximately one
part of liquid fraction-1 and two parts of liquid fraction-2. No
enzymes are added to the mixed sugar hydrolysis. Enzymes are a part
of liquid fraction-2. The enzymes added to the fiber hydrolysis and
which stay in solution will follow the liquid fraction-2 after
solid liquid separation of the fiber hydrolysis.
[0474] Experimental work was set-up to prove if there is a
difference in the efficiency of xylo-oligomer conversion in MSH
(without fibers) and in MSH including fibers (C5 by-pass
process).
[0475] Standard fiber hydrolysis was conducted in the free fall
reactor in 10 kg scale. After 72 hours of fiber hydrolysis, half of
the slurry was pressed and the other part was kept as a slurry. The
filtrate (liquid fraction-2) and the slurry were transferred to
individual shake flasks and liquid fraction-1 was added to all the
shake flasks. The MSH was conducted for 96 hours at standard
conditions.
Results
[0476] In FIG. 12, it is seen that the MSH added liquid fraction-1
(without fibers) is giving a similar xylan conversion as the MSH
added slurry (with fibers). These data indicate that most of the
relevant enzymes (xylanases) in Cellic.RTM. Ctec3 are following the
filtrate (liquid fraction-2) after fiber hydrolysis.
[0477] In the MSH, approximately 40% of the xylan is converted in
to monomeric xylose at time zero. Very fast (<10 h) 60% of
xylose potential is converted to xylose. After 10 hours, the
conversion rate is very slow. At 48 h of MSH, 63-67% of conversion
is obtained. By adding high amounts of Cellic.RTM. Ctec3, xylan
conversion degrees of up to 90% in 48 h were obtained (see e.g.
FIG. 13). It is further proven in spiking experiments (data not
shown) that monomeric sugars (glucose and xylose) are not
inhibiting xylan conversion, but pretreatment inhibitors and
oligomer concentration in the mixed sugar hydrolysis have a
significant inhibiting effect on xylan conversion.
Conclusion
[0478] The MSH is equally efficient with and without fibers,
meaning that enzymes important for the hydrolysis of hemicellulose
fragments and xylo-oligomers are soluble and following the water
phase.
Example 5--Dose response in Mixed Sugar Hydrolysis
[0479] Fiber hydrolysis was conducted in the free fall reactor in
10 kg scale. After 72 hours of fiber hydrolysis, the slurry was
pressed. The filtrate (liquid fraction-2) and the liquid fraction-1
was heated to 80.degree. C. for 20 min. to deactivate the enzyme
activity. 66 g of the filtrate (liquid fraction -2) from the press
was transferred to 12 shake flasks and 33 g liquid fraction-1 was
added to all the shake flasks. Different amount of Cellic.RTM.
CTec3 was added and the MSH was conducted for 48 hours at standard
conditions. The concentration in the mixture was measured and the
conversion for no enzyme addition in heat-treated liquid was
calculated. A MSH with not heated liquids and no enzyme addition
was conducted together with the other shake flasks showing the
conversion due to enzymes following the filtrate from the fiber
hydrolysis.
Results
[0480] FIG. 13 shows the total xylose conversion as a function of
enzyme dosage. If all enzyme added to the fiber hydrolysis had been
added to the MSH, it would have corresponded to 240 g Cellic.RTM.
Ctec3/kg sugar. The result without adding active enzyme indicates
that only a relatively low part of the enzyme in the filtrate
stream is active. Nevertheless, it can also be seen that with a
sufficiently high enzyme concentration and hydrolysis time, total
xylan conversion of up to 90% can be achieved.
[0481] As high enzyme concentration leads to almost complete and/or
faster conversion of xylan, recycling of enzymes in MSH could be of
great advantage. Ultra-filtration (UF) is a normal unit operation
for recovering enzymes from fermentation broth. In this V2.X
process, UF could recover enzymes after MSH and recycle them to the
MSH. It will lead to high enzyme concentration in MSH over time,
which will improve the hydrolysis of xylo-oligomers.
[0482] It is also thought that only some enzyme activities are
missing due to instability and thus loss of activity during the
first 100 hours of reaction or adsorption to the fibers or soluble
compounds such as organic degradation products or carbohydrates.
Addition of single activities as for example .beta.-xylosidase can
thus lead to a great increase in conversion and could be
advantageous compared to adding large amounts of enzyme
mixtures.
Conclusion
[0483] Enzymatic hydrolysis of oligomeric hemicellulose is possible
in the liquid fractions from the V2.X process. A conversion of 67%
is achieved by hydrolysis of enzymatic activity transferred through
the filtrate to the mixed sugar hydrolysis. However, up to 90%
could be achieved by adding more enzyme, Cellic.RTM. CTec3 or other
commercial enzyme mixtures or single activities such as
.beta.-xylosidase or others. Increased conversion could also be
obtained by recirculating the enzyme for example through
up-concentration by ultra-filtration.
Example 6--Addition of .beta.-xylosidase in MSH
[0484] It is believed that addition of .beta.-xylosidase will
increase the xylan conversion significantly, such as to at least 80
or 90% in the MSH.
[0485] The fiber hydrolysis is conducted in the free fall reactor
in 10 kg scale using 75 g CTec3/kg glucan. The slurry is pressed
after 72 hours of fiber hydrolysis. Thereafter, 66 g of the
filtrate (liquid fraction-2) and 33 g liquid fraction-1 are
transferred to 27 shake flasks (three groups in triplicates). In
the first group, .beta.-xylosidase is added in a concentration
corresponding to 1, 5, 10, 20 and 40% of the "total" enzyme protein
added with CTec3 to the fiber hydrolysis. In the second group,
CTec3 is added in a concentration corresponding to 10 and 40% of
CTec3 added to the fiber hydrolysis. The third group is a control
group without extra enzymes addition. The concentration of xylose
in the mixture is measured by HPLC and the xylan conversion
calculated for each treatment.
[0486] The .beta.-xylosidase can e.g. be from Bacillus pumilus,
such as a high purity recombinant .beta.-xylosidase obtainable from
Megazyme (EC 3.2.1.37; CAZy Family: GH43; CAS: 9025-53-0; in 3.2 M
ammonium sulphate; supplied at .about.75 U/mL, with a specific
activity of .about.18 U/mg (35.degree. C., pH 7.5 on
p-nitrophenyl-.beta.-D-xylopyranoside).
[0487] Samples with extra .beta.-xylosidase reveal a significantly
higher xylan conversion, such as at least 80 or 90% xylan
conversion after MSH, in contrast to the control without enzyme
and/or those with CTec3-addition, with a xylan conversion at around
66%.
Example 7--Comparison of Fermentation Substrates from V2 and V2.X
Method
[0488] The fermentation process in the V2.X process (see e.g. FIGS.
1 and 4) differs from the fermentation process in the C5 by-pass
process (V2), process scheme (2) (see e.g. FIG. 3), inter alia, in
the way that the fermentation is fed from more than one substrate
(hydrolysate from fiber cake hydrolysis and mixed sugar
hydrolysis). Furthermore, the fiber cake hydrolysate (Slurry C6+C5
product can be subjected to a further solid/liquid separation step.
In the C5 by-pass process there is only one substrate (see FIG. 3)
and the fermentation needs dilution with water in the start of the
fed batch due to high concentrations of acetic acid and other yeast
inhibiting substances as furfural. Otherwise, the time needed for
e.g. sugar to ethanol conversion would be prolonged significantly.
The composition of the hydrolysates for fermentation in the V2.X
process can be seen in Table 8.
TABLE-US-00007 TABLE 8 Composition of hydrolysates from mixed sugar
hydrolysis MSH and fiber cake hydrolysis (see e.g. FIG. 1, steps
(e), and step (f), respectively). Hydrolysate from step f)
Hydrolysate [g/kg wet] from step e) Glucose 97 57 Xylose 20 44
Acetic acid 2.3 9.7 Furfural 0.3 1.2 5-HMF 0.1 0.3 5-HMF:
5-(hydroxymethyl)furfural
[0489] As can be seen from Table 8, the hydrolysate from step (f)
(hydrolysate-1) contains significantly lower inhibitor
concentrations (acetic acid, furfural and 5-HMF) than the
hydrolysate from step (e) (hydrolysate-2).
[0490] Surprisingly and unexpectedly, the inventors have realised
that the two hydrolysates with different inhibitor concentration
can be used in a novel and advantageous fermentation process.
Commonly, at the beginning of a fermentation, the hydrolysate is
diluted with water in order to reduce the inhibitor concentration
to an acceptable level. According to the present invention, a
hydrolysate, which is low in inhibitor--e.g. the Slurry C5/C6
product or the liquid C5/C6 product obtained after a solid liquid
separation of said Slurry C5/C6 product--, can be used in an
initial phase of a microbial fermentation, usually a batch
fermentation. In this way, dilution water can be avoided or reduced
(if some dilution is still necessary), fermentation time can be
reduced, and production costs can be reduced, as less water needs
to be removed from the fermentation product, apart from the
afore-mentioned timesavings. It is also conceivable that a faster
fermentation, as provided e.g. according to the present invention,
will reduce the risk of contaminations, i.e. growth of undesired
microorganisms, resulting in lower fermentation product yields.
[0491] When performing a fermentation, a fed batch set-up may
provide one or more of the following benefits:
[0492] (1) Furan and/or other inhibitory compounds inhibit
different microorganisms including yeast, and consequently, these
need to be controlled and/or reduced to a suitable low level to
improve growth and/or fermentation product formation, such as yeast
growth and EtOH production. When using a fed-batch phase approach
this can be achieved, as yeast removes e.g. furans present in the
initial batch phase. During the fed batch phase yeast continuously
removes furans, so the detected level is very low or even close to
zero.
[0493] (2) Acetic acid is another inhibitor, and by choosing an
initial batch phase with a low level, the production strain will
have an easier start, beginning to grow and produce product
faster.
[0494] (3) In fed batch it is possible to control the feed addition
so that the concentration of glucose is kept below approx. 10 g/kg
wet, improving the conversion of xylose in C5 GMO yeast.
[0495] In all cases, it may also be important to choose the optimal
start volume in the initial batch phase compared to the total
volume, and an optimal (small) amount of yeast inoculum.
[0496] An advantage of having two hydrolysate qualities in term of
inhibitor concentration, as in Version 2.X is, that it is possible
to conduct fed batch fermentations without dilution in the initial
batch phase, while still being able to convert essentially all
xylose added in a suitable time frame, even at a very high acetic
acid concentration (such as of app. 10 g/kg), and also with a
relatively high overall furfural concentration (such as around 1
g/kg wet hydrolysate).
Example 8--Improved Fermentation in V2 Process
[0497] For a fermentation in the V2 process, all C5 bypass liquid
is added to the "post hydrolysis", thereby providing only a single
substrate for fermentation (see e.g. FIG. 3). Terms related to
"post hydrolysis" used in some examples are believed to be
corresponding to mixed sugar hydrolysis (MSH). Likewise, terms
related to "C5 bypass" or "C5 bypass liquid faction" are believed
to be corresponding to "liquid fraction 1" or "first liquid
fraction". Commonly, it is necessary to dilute the post hydrolysis
substrate with water in the "initial batch phase" of a fed batch
fermentation, in order to reduce the concentration of inhibitors,
such as to provide an efficient and/or reliable fermentation, such
as in terms of growth of microorganism and/or fermentation product
yield. Usually, fermentations are yeast fermentations, aiming at
production of 2G EtOH, however, it is believed that other
microorganisms can be used as well, thus also providing different
fermentation products.
[0498] Surprisingly and unexpectedly, the inventors have realised
that also the V2 process may be adapted in view of the above
findings related to the hydrolysates or product streams with
different inhibitor concentrations. See FIG. 14 for an example of a
principal set-up of a process for such an improved fermentation.
Consequently, in order to avoid (or reduce) dilution in the start
of fermentation, it is believed to be possible to use the
hydrolysate from the first hydrolysis (e.g. from step (c)), as the
material to start the fermentation. This hydrolysate is much lower
in yeast inhibitor concentration than the Post Hydrolysate. Once
the amount of hydrolysate from step (c) which is needed for the
initial batch phase of the fermentation has been removed, the
remaining hydrolysate can be mixed with C5 bypass liquid fraction
to ensure hydrolysis of C5 oligomers present in C5 liquid. The post
hydrolysis step will then have a smaller fraction of hydrolysate
from step (c), compared to C5 liquid than in the original set-up.
The GMO yeast indicated in FIG. 14 is optional, other suitable
microorganisms could be used as well, thus also allowing for
provision of other fermentation products than e.g. alcohol/EtOH.
Furthermore, it is believed that such a process will work reliable
with different DM concentrations, and different xylan numbers. A
scheme of a two-step fermentation is seen in FIG. 15. An initial
fermentation is conducted using only a fraction, such as e.g.
around 5%, 10% or 20% of the fermenter volume, followed by second
fermentation, whereby the fermenter is filled. The first
fermentation can e.g. be a batch fermentation, and the second
fermentation a fed-batch fermentation.
[0499] The proportion between C5 liquid fraction and hydrolysate
from step (c) will be higher in the post hydrolysis (see FIG. 3) in
the proposed improved V2 process. It has been tested, if the
hydrolysis in step (d) would be negatively affected. Below is a
description of how this was tested, and that the hydrolysis is not
negatively affected. Results are shown in FIG. 16.
Materials and Methods
[0500] The fiber hydrolysis (step (c)) were conducted in a vertical
pilot reactor in 240 kg scale using 75 g CTec3/kg glucan with a dry
matter of 22% suspended solids. After 117 hours of fiber
hydrolysis, the slurry was pressed. The filtrate and the C5 liquid
fraction was mixed in different ratios 1:2, 1:1 and 2:1 in shake
flasks with at total volume of 100 gram. The Post Hydrolysis was
conducted in shaking incubators for 48 hours at standard
conditions; pH 5.0-5.3; 50.degree. C. The concentration of xylose
in the mixture was measured by HPLC and the xylan conversion of C5
liquid fraction was calculated for each ratio. To calculate the
xylan conversion of C5 liquid fraction, it was assumed that the
filtrate from hydrolysis step (c) did not contribute to the
increase in xylose concentration as a function of time. FIG. 16
shows changes in xylan conversion at increasing proportions of C5
liquid in the post hydrolysis. Filtrate is the liquid fraction
after the hydrolysis step (c). Only very limited effects on
hydrolysis efficiency are seen in FIG. 16 (blending 1:1 is a far
higher proportion of C5 liquid than would be the case for the
improved fermentation setup).
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